Biomedical subjects

Shao-xi Cai

Publications and source records attributed to Shao-xi Cai.

At least 19 recordsLinked to original sources

PubMed2006

Effects of serum of the rats ventilated with high tidal volume on endothelial cell permeability and therapeutic effects of ulinastatin.

BACKGROUND: With the widespread use of ventilators in treating critically ill patients, the morbidity of ventilator-induced lung injury (VILI) is increasing accordingly. VILI is characterized by a considerable increase in microvascular leakiness and activation of inflammatory processes. In this study we investigated the effects of inflammatory mediators in VILI rat serum on endothelial cytoskeleton and monolayer cellular permeability, as well as the therapeutic effect of ulinastatin, to explore the pathogenesis and the relationship between biotrauma and lung oedema induced by VILI. METHODS: Thirty healthy male Sprague-Dawley rats were randomly divided into three groups: group A (normal tidal volume ventilation), group B (high tidal volume ventilation) and group C (high tidal volume ventilation plus ulinastatin). The serum of each rat after ventilation was added to endothelial cell line ECV-304 medium for two hours to observe the effects of serum and/or ulinastatin on endothelial fibrous actin and permeability. RESULTS: Compared to rats ventilated with normal tidal volume, serum of rats ventilated with high tidal volume caused a striking reorganization of actin cytoskeleton with a weakening of fluorescent intensity at the peripheral filament bands and formation of the long and thick stress fibres in the centre resulting in endothelial contraction and higher permeability. Prior treatment with ulinastatin lessened the above changes significantly. The changes of permeability coefficient of endothelial permeability after group A, B or C rats serum stimulation were (6.95 +/- 1.66)%, (27.50 +/- 7.77)% and (17.71 +/- 4.66)% respectively with statistically significant differences (P < 0.05) among the three groups. CONCLUSIONS: The proinflammatory mediators in the serum of the rats given high tidal volume ventilation increases endothelial permeability by reorganizing actin cytoskeleton, and pretreatment with ulinastatin lessens the permeability by inhibiting of proinflammatory mediators.

Actins↗

PubMed2006

Anti-inflammatory effects of a novel peptide designed to bind with NF-kappaB p50 subunit.

AIM: To explore the anti-inflammatory effects of a novel peptide designed to bind with the NF-kappaB p50 subunit. METHODS: The affinity of the peptide binding with p50 was measured with a biosensor. Levels of tumor necrosis factor-alpha(TNF-alpha) and interleukin 6 (IL-6) from a human acute monocytic leukemia cell line (THP-1) treated with lipopolysaccharide (LPS) were measured using the ELISA method. In vivo anti-inflammatory effects of the peptide were tested with phorbol myristate acetate (PMA)-induced ear edema and zymosan A-induced peritonitis in mice. RESULTS: The peptide has the ability to interact with the NF-kappaB p50 subunit and can effectively inhibit TNF-alpha and IL-6 production in the THP-1 cell line, PMA-induced ear edema and zymosan A-induced peritonitis in mice. CONCLUSION: The peptide may have therapeutic potential for the treatment of local acute inflammation.

Animals↗

PubMed2006

[Construction of subtracted cDNA library by suppression subtractive hybridization for differentially expressed genes in eosinophils from asthma patients].

OBJECTIVE: To construct a subtracted cDNA library of differentially expressed genes in eosinophils from asthma patients. METHODS: Suppression subtractive hybridization (SSH) was used to isolate the cDNA fragments of differentially expressed genes in the eosinophils of asthma patients before and after treatment. The cDNA fragments were directly inserted into T/A cloning vector to establish the subtractive library, followed by amplification of the library through E. coli transformation with calcium chloride and screening of blue and white clones of the transformants. One hundred positive bacterial clones were randomly picked and identified by colony PCR. RESULTS: The amplified library contained more than 3,000 positive bacterial clones. Analysis of the randomly selected 100 white clones by PCR showed that 90% of the clones contained 100-500 bp inserts, which might be the cDNA fragments of differentially expressed genes in eosinophils of asthma patients before treatment. CONCLUSION: A subtracted cDNA library of differentially expressed genes in the eosinophils of asthma patients before and after treatment is constructed successfully by SSH and T/A cloning techniques, which lays a solid foundation for screening and cloning new specific differentially.expressed genes in the eosinophils of asthma patients.

Asthma↗

PubMed2006

Expression of thioredoxin-binding protein-2/vitamin D3 upregulated protein-1 in peripheral blood eosinophils of asthma patients.

OBJECTIVE: To investigate the expression of vitamin D3 upregulated protein-1 (VDUP1) in peripheral eosinophils at different stages of asthma and its correlation with clinical manifestations of asthma. METHODS: Fourteen normal volunteers and 51 mild to moderate asthma patients, including 16 untreated patients with asthma attack and 35 with asthma remission by continuously corticosterone inhalation. The symptom severity and pulmonary function indices were evaluated and induced sputum eosinophil counts and blood eosinophil count measured. VDUP1 and beta-actin gene fragments were amplified simultaneously by RT-PCR from the total RNA of peripheral eosinophils, and 10 microl of the RT-PCR product underwent agarose gel electrophoresis and the VDUP1/beta-actin ratio was obtained by Gel-Pro software. RESULTS: VDUP1/beta-actin ratio significantly decreased in untreated patients with asthma attacks in comparison with normal volunteers (0.314+/-0.242 vs 0.532 +/-0.279) but not in patients with asthma remission (0.612+/-0.381). In the former patients, a positive correlation of VDUP1/beta-actin ratio was found with FEV1.0% (r=0.587, P=0.046) and %PEF (r=0.563, P=0.033), whereas an inverse one observed with sputum eosinophil count (r=-0.436, P=0.049). CONCLUSION: VDUP1 expression in the eosinophils correlates to eosinophil activation and may influence the disease severity of asthma patients.

Actins↗

PubMed2005

[Angiotensin II and aldosterone stimulate alpha1-(I) procollagen mRNA expression in hepatic stellate cells via activation of ERK1/2 and AP-1].

OBJECTIVE: To investigate the signal transduction mechanism underlying the effects of angiotensin II (Ang II) and aldosterone (Aldo) on the signal passageway of active protein-1 (AP-1). METHODS: In vitro, Hepatic stellate cells (HSCs) of the line HSC-T6 were cultured and treated with Ang II or Aldo, the principal effector molecules of the renin-angiotensin-aldosterone system (RAAS) for 10, 30, 60, 120, and 180 minutes respectively. The protein expression of phospho-P42/44 was detected by Western blotting. In addition, HSC-T6 cells were preincubated for 60 min with U0126, an inhibitor of MAPK/ERK kinase, irbesartan, an AT-1 receptor blocker, N-acetylcysteine (NAC), antioxidant, angiotensin converting enzyme inhibitor (ACEI), or tumor necrosis factor alpha (TNFalpha) prior to exposure to Ang II or Aldo. Then the protein expression of phospho-P42/44 was measured by Western blotting. The DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, the mRNA expression of alpha1 (I) procollagen was detected. RESULTS: The levels of phopho-ERK1/2 protein increased after the treatment of Ang II and Aldo at all time points and both peaked 10 minutes after (both P < 0.01). The levels of phopho-ERK1/2 protein of the irbesartan + Ang II and U0126 + Ang II groups were significantly lower than that of the Ang II group (both P < 0.01). The level of phopho-ERK1/2 protein of the Ang II group was lower than that of the TNFalpha group, however, was especially significantly lower than that of the Ang II + TNFalpha group (P < 0.01). The level of phopho-ERK1/2 protein of the U0126 + Aldo group was significantly lower than that of the Aldo group (P < 0.01). The phopho-ERK1/2 protein level of the NAC + Aldo group was not significantly different from that of the Aldo group (P > 0.05). The phopho-ERK1/2 protein level of the Aldo group was lower than that of the TNFalpha group, however, was especially significantly lower than that of the Aldo + TNFalpha group (P < 0.01). The AP-1 DNA binding protein increased after the treatment of Ang II and peaked 30 min after. U0126, irbesartan, and NAC, as well as ACEUI, significantly inhibited the increased AP-1 DNA binding activity induced by Ang II. The AP-1 DNA binding protein increased after the treatment of Aldo and peaked twice, 30 min and 240 min after. U0126 and NAC significantly and NAC partly inhibited the increased AP-1 DNA binding activity induced by Aldo. CONCLUSION: Stimulation of HSC by Ang II and Aldo results in activation of AP-1 via ERK1/2 pathway leading to up-regulation of AP-1 target gene alpha1 (I) procollagen mRNA expression.

Aldosterone↗

PubMed2005

Identification of human dopamine D1-like receptor agonist using a cell-based functional assay.

AIM: To establish a cell-based assay to screen human dopamine D1 and D5 receptor agonists against compounds from a natural product compound library. METHODS: Synthetic responsive elements 6 cAMP response elements (CRE) and a mini promoter containing a TATA box were inserted into the pGL3 basic vector to generate the reporter gene construct pCRE/TA/Luci. CHO cells were co-transfected with the reporter gene construct and human D1 or D5 receptor cDNA in mammalian expression vectors. Stable cell lines were established for agonist screening. A natural product compound library from over 300 herbs has been established. The extracts from these herbs were used for human D1 and D5 receptor agonist screenings. RESULTS: A number of extracts were identified that activated both D1 and D5 receptors. One of the herb extracts, SBG492, demonstrated distinct pharmacological characteristics with human D1 and D5 receptors. The EC(50) values of SBG492 were 342.7 microg/mL for the D1 receptor and 31.7 microg/mL for the D5 receptor. CONCLUSION: We have established a cell-based assay for high-throughput drug screening to identify D1-like receptor agonists from natural products. Several extracts that can active D1-like receptors were discovered. These compounds could be useful tools for studies on the functions of these receptors in the brain and could potentially be developed into therapeutic drugs for the treatment of central nervous system diseases.

Animals↗

PubMed2005

[Study of genes that differentially expressed in eosinophils of patients with asthma by suppression subtractive hybridization technology].

OBJECTIVE: To study genes that differentially expressed in peripheral blood eosinophils of patient with asthmatic. METHODS: Total RNA extracted from eosinophils of patient at the time of exacerbation was taken as the tester and the total RNA from eosinophils of the same patient after improvement as driver. The ds cDNA was synthesized by SMART PCR cDNA Synthesis technology. The differentially expressed genes were obtained by suppression subtractive hybridization (SSH) technology and were further identified by Southern blot. RESULTS: Twelve differentially expressed genes including transformation growth factor beta activated kinase like (TAK1L) protein, cGMP gated channel protein were obtained. CONCLUSION: Increased expression of these genes were involved in the regulation of proinflammatory response, signal transduction, energy metabolism and cell apoptosis, which may help to clarify the molecular mechanism of eosinophils in asthma and may provide the theoretical base for finding out the new medicines towards eosinophils.

Asthma↗

PubMed2005

[Effect of different tidal volume ventilation on rat bronchial and alveolar epithelial cell apoptosis].

OBJECTIVE: To investigate the changes in bronchial and alveolar epithelial cell apoptosis after mechanical ventilation with different tidal volumes and explore the mechanism and significance of cell apoptosis in ventilation-induced lung injury. METHODS: Thirty healthy male Sprague-Dawley rats were randomly divided into groups A, B and C to receive tracheotomy under anesthesia for ventilation with different tidal volumes (VT) for 4 h (VT of 10 ml/kg in group A, 20 ml/kg in group B and 40 ml/kg in group C). Lung histopathology was assessed after the ventilation and compared between the 3 groups. The severity of lung injury and bronchial epithelial edema was evaluated, and the extent and location of cell apoptosis in the bronchus and lung tissues were studied by transferase d-UTP end-labeling (TUNEL) assay and immunohistochemistry. RESULTS: After mechanical ventilation for 4 h, lung injury and bronchial epithelial edema increased with the increment of VT. By TUNEL assay, the apoptosis index of both alveolar and bronchial epithelial cells was found to increase with VT. Immunohistochemical study identified enhanced caspase-3 expression in the cytoplasm and nuclei of airway and alveolar epithelial cells and vascular endothelial cells in rats with high VT ventilation, and the enhancement was especially obvious in the bronchial epithelial cells. CONCLUSION: Mechanical ventilation with different VTs causes consistent changes in histopathology, cell apoptosis and caspase-3 expression in rats, which support the presumption that cell apoptosis plays an important role in the pathogenesis of high VT ventilation-induced lung injury.

Animals↗

PubMed2005

[Method for human peripheral blood eosinophil isolation for patch-clamp study].

OBJECTIVE: To establish a rapid and economic method for isolating human peripheral blood eosinophils with high viability for patch-clamp studies and investigate the electrophysiological properties of Ca(2+)-activated K(+) channel of the isolated cells. METHODS: Peripheral blood eosinophils were isolated by modified discontinuous Percoll density gradient centrifugation, and the electric currents in the single Ca(2+)-activated K(+) channels of the cells were recorded using patch-clamp technique with cell-attached configuration. RESULTS: The purity of the eosinophils from healthy donors reached (90.5+/-1.6)%, with a viability rate over 99% and recovery rate of (48.2+/-6.9)%. The isolated cells were morphologically intact, from which Ca(2+)-activated K(+) channel activity could be detected. CONCLUSION: The peripheral blood eosinophils isolated using this rapid, simple and highly efficient method are characterized by high purity and viability without obvious cellular injuries, which are ideal for patch-clamp studies.

Cell Separation↗

PubMed2005

[Dyuamical studies on metabolic chemistry of lignans from seeds of Arctium lappa].

OBJECTIVE: To study the metabolic chemistry and pharmaco-dynamics characters of ligan from seeds of Arctium lappa. METHOD: HPLC method was used in the study. The analysis was carried out on C18 column. The mobile phase was CH3CN-0.05% H3PO4 (36:64) with flow-rate at 0.6 mL x min(-1) and wave-length of 210 nm. The column temperature was kept at 25 degrees C. RESULT: The results indicated that the ligan was detected in plasma and the main organs 5 min after po. The main metabolic production in plasma was arctigenin. In addition, arctigenin and an unknown product were found in metabolic production in the organs. CONCLUSION: The method was stable,simple and reproducible. It can be used to determine the metabolic product of the ligan. The metabolic chemistry of ligan in plasma was obviously different from that in the main organs.

Animals↗

PubMed2005

[Cloning and expression of human interleukin 24 gene and study on its activity of inducing tumor cells apoptosis in vitro].

AIM: To clone and express IL-24 gene in E.coli and to study its antitumor activity to induce apoptosis of tumor cells after purification and refolding. METHODS: The total RNA was extracted from peripheral blood mononuclear cells (PBMC) and induced by ConA. The human IL-24 gene was amplified by RT-PCR before subcloned into expression vector pET32a(+) and expressed in E. coli. The fusion protein was identified by Edman and purified in chelating sepharose fast flow chromatography and refolding. The activity of inducing tumor cells apoptosis was observed by MTT colorimetry and morphological study in vitro. RESULTS: The obtained human IL-24 gene was identical with that included in GenBank. The recombinant IL-24 could be expressed in E. coli by IPTG induction as inclusion body (35 kD) with purity over 95%. The apoptosis of MCF-7 cells was induced by Trx-IL-24 protein. CONCLUSION: The fusion protein can be highly expressed in E. coli and the obtained protein with high purity can induce tumor cells apoptosis.

Apoptosis↗

PubMed2005

[Aldosterone stimulates alpha1-(1) procollagen mRNA expression in HSC via activation of ERK1/2 and AP-1].

OBJECTIVE: It has been known that the intrahepatic renin-angiotensin-aldosterone system (RAAS) plays a key role in the fibrogenesis in livers. Aldosterone (Aldo), the principal effector molecule of the RAAS, exerts local effects on cell growth and fibrogenesis. However, the signal transduction mechanisms underlying the effects of Aldo on hepatic fibrogenesis remain to be fully elucidated. The present study aims to investigate the signal transduction mechanism underlying the effects of Aldo on the signal passageway of active protein-1 (AP-1). METHODS: In vitro, HSCs-T6 cell line was treated with Aldo for 10 min, 30 min, 60 min, 120 min and 180 min, and protein expression of Phospho-P42/44 was detected by Western blot. In addition, HSCs-T6 cell line was preincubated for 60 min or not with U0126 (an inhibitor of the MAPK/ERK kinase), and also with antioxidant-N-acetylcysteine (NAC) prior to exposure to Aldo for the indicated times. Protein expression of Phospho-P42/44 was measured by Western blot. DNA biding activity of AP-1 was analyzed by electrophoretic gel mobility shift assay (EMSA). By means of RT-PCR, expression of alpha1(1) procollagen mRNA was detected. RESULTS: Aldo stimulated HSC via extracellular signal-regulated kinase (ERK1/2) pathway. Time course experiments showed that Aldo induced Phospho-P42/44 expression, which was abrogated by U0126, reaching a maximum at 10 minutes, and then declined progressively. NAC inhibited the Phospho-P42/44 expression. EMSA showed that stimulation of HSC by Aldo markedly increased AP-1 DNA binding activity. U0126 markedly reduced AP-1 DNA binding activity induced by Aldo; NAC partly decreased AP-1 activity induced by Aldo. Aldo up-regulated expression of alpha1(1) procollagen mRNA, which was attenuated by U0126 and NAC. CONCLUSION: Stimulation of HSC by Aldo results in activation of AP-1 via ERK1/2 pathway, leading to up-regulation of AP-1 target gene alpha1(1) procollagen mRNA expression.

Aldosterone↗

PubMed2005

[Effect of doxofylline on calcium-activated potassium channels in human peripheral blood eosinophils in asthma].

OBJECTIVE: To study the effects of doxofylline on calcium-activated potassium (K(Ca)) channels of human peripheral blood eosinophils in asthma. METHODS: Peripheral blood eosinophils from patients with asthma were isolated and divided equally into two groups, a control group and a doxofylline incubated group. The data were recorded using cell-attached configuration of patch-clamp technique and the kinetic changes of K(Ca) channels activated by 0.2 micromol/L platelet activating factor (PAF) were compared. RESULTS: As compared with the control group, the open probability of the K(Ca) channels decreased from 0.135 +/- 0.021 to 0.044 +/- 0.018, the open time from (5.75 +/- 0.40) ms to (2.39 +/- 0.13) ms, while the close time increased from (2.17 +/- 0.50) ms to (23.73 +/- 2.50) ms in the doxofylline incubated group. The differences were significant between the two groups (all P < 0.05). CONCLUSION: Doxofylline could decrease the open probability of the channels as a result of both the shortening of open period and the prolongation of close time.

Adolescent↗

PubMed2004

Loss of C-terminal alpha-helix decreased SDF-1alpha-mediated signaling and chemotaxis without influencing CXCR4 internalization.

AIM: To investigate the possibility that a novel alpha-helix-defective mutant of stromal cell-derived factor-1alpha (SDF-1alpha) (SDF-1/54R) acts as an antagonist of CXC chemokine receptor 4 (CXCR4). METHODS: According to the genetic sequence of natural SDF-1alpha, a recombinant alpha-helix-defective mutant of SDF-1alpha was designed and some biologic characteristics of this mutant were demonstrated. The migration of Jurkat cells was assessed with chemotactic assay. ERK phosphorylation was analyzed by Western blot with a specific anti-phospho-ERK1/2 antibody. Intracellular calcium influx was examined by flow cytometer with a calcium indicator dye Fluo-3AM. The CXCR4 on the cell surface was detected by flow cytometer with a PE conjoined anti-human CXCR4 antibody. RESULTS: Compared with native SDF-1alpha, SDF-1/54R displayed apparent decrease in chemotactic ability, ERK1/2 activation, and intracellular calcium influx in Jurkat cells. However, the binding to CXCR4 and inducing CXCR4 internalization of SDF-1/54R did not change outstandingly. Moreover, a competitive inhibitory effect of SDF-1/54R on the migration of Jurkat cells induced by native SDF-1alpha was confirmed. CONCLUSION: Alpha-helix-defective mutant of SDF-1alpha, SDF-1/54R that remained both the N-terminus and the central beta-sheet region, decreased SDF-1alpha-mediated signaling and chemotaxis but did not influence CXCR4 internalization, which suggested that SDF-1/54R might be developed as an anti-CHIV inhibitor with high biological potency and low side-effect.

Calcium↗

PubMed2004

[The expression of lung nitric oxide synthase in rat exposed to silica].

OBJECTIVE: To evaluate the time-effect of silica on the expression of lung tissue nitric oxide synthase (NOS) in early inflammatory damage stage of silicotic rat. METHODS: Animal models were established by direct tracheal instillation of silica into rat lungs. Total NOS and induced NOS (iNOS) activities in bronchoalveolar lavage fluid (BALF) were assayed. The expression of iNOS protein in paraffin-embedded lung sections with Streptavidin/peroxidase (SP) immunohistochemistry were measured by tissue microarray and Image-Pro Plus. RESULTS: Most of the expression of iNOS was in the cytoplasm of macrophages and neutrophils. iNOS integrated optical density (IOD) of lung tissue increased 1.47 x 10(5) and 2.73 x 10(5) more respectively in silicatreated rats 3, 7 days after exposure than in control rats (P < 0.05), and decreased 1.11 x 10(5) more 28 days after exposure (P < 0.01). The activities of iNOS in BALF increased by 0.86, 1.89 and 0.92 U/ml respectively 3, 7, 14 days after exposure (P < 0.05 or P < 0.01). The activities of total NOS in BALF increased by 1.43, 2.05, 2.61 and 2.19 U/ml respectively 1, 3, 7, 14 days after exposure (P < 0.05 or P < 0.01). CONCLUSION: After silica instillation, the iNOS-positive cells in rat lung tissue were mostly macrophages and neutrophils. There is a parabolic changing trend in the level of expression of lung iNOS during 1 - 28 day exposure to silica.

Animals↗

PubMed2004

[Cloning of differentially expressed genes of eosinophils from asthmatic patients by suppression subtractive hybridization].

OBJECTIVE: To explore the molecular mechanism of eosinophils for its role in bronchial asthma. METHODS: The total RNA extracted from the eosinophils of patients during the onset of asthma was used as the tester and the total RNA obtained after treatment served as the driver. cDNA suppression subtractive hybridization (SSH) was performed using the protocols described in the Clontech SMART PCR cDNA Syn thesis Kit and PCR-Select cDNA Subtraction Kit. The subtracted cDNA was then inserted into T vector to generate subtracted cDNA library. Clones of the subtracted cDNA library were screened by hybridization and the insert sequence of the positive clones was compared with the sequence in the GenBank. RESULTS: A total of 400 clones selected from the subtracted cDNA library were amplified by PCR and about 85% of these clones contained inserts. Six differential cDNA fragments were acquired after two differential screening. These genes were involved in the regulation of proinflammatory response, signal transduction, energy metabolism and cell apoptosis. CONCLUSION: Differentially expressed genes of the eosinophils during the onset and the remission stage of bronchial asthma can be effectively cloned by SSH, which provides a solid foundation for clarifying the molecular mechanism of eosinophils in asthma and a theoretical base for clinical treatment and prevention of asthma.

Asthma↗

PubMed2004

[Super SMART cDNA synthesis technology for amplifying small amount of total RNA of peripheral blood eosinophils from asthma patients].

OBJECTIVE: To amplify double-strand cDNA from small amount of total RNA of eosinophils from asthma patients by Super SMART cDNA synthesis technique. METHODS: The eosinophils were purified from the peripheral blood of asthma patients before and after treatment by Percoll gradient centrifugation, from which the total RNA was extracted using TRIzol kit. First-strand cDNA synthesis and double-strand cDNA amplification were performed using Super SMART cDNA synthesis technique. The quality of the obtained cDNA was evaluated by gradient cDNA electrophoresis, and the amplification efficiency determined by cDNA quantification. RESULT: From 20 ng total RNA, 7.155 microg and 6.568 microg of the tester and driver double- strand cDNAs respectively were obtained successfully, and the result of electrophoresis indicated high quality and purity of the cDNA acquired. CONCLUSION: Super SMART cDNA synthesis technique can effectively amplify high-quality double-strand DNA from a very small amount of total RNA, which may facilitate the exploration of the mechanism of asthma from the genetic level.

Asthma↗

PubMed2003

[Integrin alpha3beta1 mediates hepatocellular carcinoma cell adhesion and chemotaxis to type IV collagen].

OBJECTIVE: To investigate the effects of Integrin alpha(3)beta(1) on the adhesion and chemotaxis of hepatocellular carcinoma (HCC) cells to type IV collagen (Col IV). METHODS: (1) HCC cells were culture and suspension of HCC cells was made. Anti-alpha(3) and Anti-beta(1) were added into the HCC cell suspension. Flow cytometry was used to determine the expression of integrin alpha(3)beta(1) on the surface of HCC. (2) 5 micro g/ml Col IV was used to coat a cell with the diameter of 25 mm. Digested HCC cells were added. Anti-alpha(3) and Anti-beta(1) of the concentrations of 5 micro g/ml and 10 micro g/ml respectively were added into the cell suspension. Before and after the addition of Anti-alpha(3) and Anti-beta(1), micropipette technique was used to measure the adhesion force of HCC on Col IV-coated surface, as function of the square of internal radius of micropipette and the critical negative pressure needed to detach a single HCC cell away from the substrate. (3) Col IV of the concentration of 600 micro g/ml was added into the dual micropipettes. Then the dual micropipettes were led towards the HCC cells. A HCC cell was made to seal the openings of the 2 micropipettes with different parts of the cell contacting Col IV in different micropipettes. The pseudopod protrusion was observed dynamically and recorded with tape recorder. The length of pseudopod was measured and plotted against the chemotactic time so as to obtain a pseudopod growth curve. RESULTS: (1) The expression rates of integrin subunit alpha(3) and beta(1) on the surface of HCC cells were 95.55% and 95.78% respectively. (2) The adhesion force of HCC cells to the 5 micro g/ml Col IV-coated surface was 932 +/- 134 (x 10(-10) N, n = 60). Upon treatment of the HCC cells with Anti-alpha(3) of the concentrations of 5 micro g/ml and 10 micro g/ml, the adhesion force decreased by 42% and 49%, to 536 +/- 122 (x 10(-10) N, n = 60) and 476 +/- 63 (x 10(-10) N, n = 60) respectively. Upon treatment of the HCC cells with Anti-beta(1) of the concentrations of 5 micro g/ml and 10 micro g/ml, the adhesion force decreased by 52% and 76%, to 449 +/- 119 (x 10(-10) N, n = 60) and 220 +/- 78 (x 10(-10) N, n = 60) respectively. (3) The length of pseudopod increased along with the chemotactic time. The pseudopod length and growth curve were almost identical in the dual micropipettes when they were filled with Col IV. When Anti-alpha(3) or Anti-beta(1) was added into one of the dual micropipettes, the HCC cell pseudopod protrusion was almost blocked completely, while the HCC cell pseudopod in the opposite micropipette became more evident. CONCLUSION: Integrin alpha(3)beta(1) is an important constituent receptor in mediating HCC cell adhesion and chemotactic pseudopod protrusion to Col IV.

Carcinoma, Hepatocellular↗