Biomedical subjects

Juan Li

Publications and source records attributed to Juan Li.

157 records · Page 9Linked to original sources

PubMed2002

Partially inverted tandem repeat isolated from pericentric region of chicken chromosome 8.

The majority of chicken repetitive sequence is nuclear-membrane-associated sequence (CNM), which resides in a large number of microchromosomes (chromosomes 11-39) and is absent from macrochromosomes 1-5, ZW, and some of the intermediate chromosomes 6-10. Two repetitive families, EcoRI/XhoI, are confined to the female-specific W chromosome. The core repeat units of the three families are 21 bp, containing (A)3-5 and (T)3-5 clusters separated by 5-7-bp sequences. In this article, we describe the isolation and initial characterization of a novel repeat family that is related to CNM/EcoRI/XhoI families. The novel family, designated as PIR, consists of multiple types of partially inverted repeat units of about 1.2, 1.4 and 1.6 kb. The PIR sequence is restricted to chicken chromosome 8, and accounts for about 3.8 mb, or 2500 copies of the 1.4-kb units, of the chicken genome. The evolution of PIR and related sequences is discussed.

Animals↗

PubMed2002

A novel gene expression control system and its use in stable, high-titer 293 cell-based adeno-associated virus packaging cell lines.

Previous attempts to establish 293cell-based stable and high-titer adeno-associated virus (AAV) packaging cell lines were unsuccessful, primarily due to adenovirus E1-activated Rep gene expression, which exerts cytostatic and cytotoxic effects on the host cells. Control of the two large AAV Rep proteins (Rep78/68) was insufficient to eliminate the adverse effects, because of the leaky expression of the two small Rep proteins (Rep52/40). However, it was unsuccessful to control Rep52/40 gene expression since its promoter is located within the coding sequence of Rep78/68. To tightly regulate all four Rep proteins by using their own promoters, we have developed a novel gene control paradigm termed "dual splicing switch," which disrupts all four Rep genes by inserting into their shared coding region an intron that harbors transcription termination sequences flanked the LoxP sites. As a result, the structure and activities of the Rep gene promoters, both p5 and p19, are not affected; however, all of the Rep transcripts are prematurely terminated and the genes were inactivated. Removal of the terminator by Cre protein reactivates the transcription of all four Rep proteins derived from their own promoters. This switch system was initially tested in the lacZ gene and a 600-fold induction of beta-galactosidase activity was observed. Using the dual splicing switch strategy, we have subsequently established a number of AAV packaging cell lines from 293 cells, which showed a normal growth rate, high stability, and more importantly, high yields of AAV vectors. Such a gene control paradigm is also useful for other viruses, e.g., autonomous parvoviruses. Finally, the high-titer 293-based AAV packaging cell lines should greatly reduce the risk of wild-type adenovirus contamination and provide a scalable AAV vector production method for both preclinical and clinical studies.

Cell Line↗

PubMed2002

Feasibility of generating adeno-associated virus packaging cell lines containing inducible adenovirus helper genes.

The adeno-associated virus (AAV) vector system is based on nonpathogenic and helper-virus-dependent parvoviruses. The vector system offers safe, efficient, and long-term in vivo gene transfer in numerous tissues. Clinical trials using AAV vectors have demonstrated vector safety as well as efficiency. The increasing interest in the use of AAV for clinical studies demands large quantities of vectors and hence a need for improvement in vector production. The commonly used transient-transfection method, although versatile and free of adenovirus (Ad), is not cost-effective for large-scale production. While the wild-type-Ad-dependent AAV producer cell lines seem to be cost-effective, this method faces the problem of wild-type Ad contamination. To overcome these shortcomings, we have explored the feasibility of creating inducible AAV packaging cell lines that require neither transfection nor helper virus infection. As a first step toward that goal, we have created a cell line containing highly inducible Ad E1A and E1B genes, which are essential for AAV production. Subsequently, the AAV Rep and Cap genes and an AAV vector containing a green fluorescent protein (GFP) reporter gene were stably introduced into the E1A-E1B cell line, generating inducible AAV-GFP packaging cell lines. Upon induction of E1A and E1B genes and infection with replication-defective Ad with E1A, E1B, and E3 deleted, the packaging cells yielded high-titer AAV-GFP vectors. Finally, the E2, E4, and VA genes of Ad, under the control of their endogenous promoters, were also introduced into these cells. A few producer cell lines were obtained, which could produce AAV-GFP vectors upon simple drug induction. Although future improvement is necessary to increase the stability and vector yield of the cells, our study has nonetheless demonstrated the feasibility of generating helper-virus-free inducible AAV producer cell lines.

Adenovirus E1A Proteins↗

PubMed2002

Study of apoptosis in human liver cancers.

AIM: To investigate the action of apoptosis in occurrence of liver carcinomas in vivo and the biological effect of Solanum lyratum Thumb on BEL-7404 cell line inducing apoptosis in vivo. METHODS: The apoptosis in the liver carcinoma was detected with terminal deoxynucl neotidyl transferase mediated dUTP nick end labelling (TUNEL); the cancer cells cultured in DMED medium were treated with extract of Solanum lyratum Thumb and observed under microscope, and their DNA was assayed by gel electrophoresis. RESULTS: In vivo apoptotic cells in the cancer adjacent tissues inceased; in vivo treatment of liver cancers with extract of Solanum lyratum Thumb could induce the cells to manifest a typical apoptotic morphology. Their DNA was fractured and a characteristic ladder pattern could be found using electrophoresis. CONCLUSION: In vivo the apoptosis of carcinomas was lower; maybe the cells divided quickly and then the cancers occurred. In the cancer adjacent tissues,the apoptosis pricked up, and in vivo Solanum lyratum Thumb could induce the apoptosis of BEL-7404 cells.

Apoptosis↗

PubMed2002

[Construction and expression of recombinant retrovirus vector carrying HBV vector].

BACKGROUND: To explore the possibility of using retrovirus vector to carry HBV vector, and to prove that replication defective HBV could be normally packaged. METHODS: Two kinds of full length of mutant HBV gene, which express dominant negative mutants, were inserted into retrovirus vector. After recombinant retroviruses were harvested, they were used to infect Hep G2 and 2.2.15 cell line. Then the expression of HBV core antigen in the Hep G2 cell was examined by immune fluorescence, and the existence of recombinant HB virion in the culture medium was examined by PCR. RESULTS: High titer of recombinant retroviruses were obtained in the culture medium of transfected PA317 cell line. Core antigen was detectable in the recombinant retrovirus infected Hep G2 cell. Recombinant HB virion was detectable in the culture medium of recombinant retrovirus infected 2.2.15 cell. CONCLUSIONS: The results suggested that recombinant retrovirus could carry HBV vector and express HBV products. When structural protein is offered by wt-HBV, the recombinant retrovirus may function as HBV vector, therefore it could be used in anti?HBV gene therapy.

Genetic Therapy↗

PubMed2002

[Anti-HBV effects of genetically engineered replication-defective HBV with combined expression of antisense RNA and dominant negative mutants of core protein and construction of first-generation packaging cell line for HBV vector].

OBJECTIVE: To explore the possibility of using HBV as a gene delivery vector, and to test the anti-HBV effects by intracellular combined expression of antisense RNA and dominant negative mutants of core protein. METHODS: Full length of mutant HBV genome, which expresses core-partial P fusion protein and/or antisense RNA, was transfected into HepG2.2.15 cell lines. Positive clones were selected and mixed in respective groups with hygromycin in the culture medium. HBsAg and HBeAg, which exist in the culture medium, were tested by ELISA method. Intracellular HBc related HBV DNA was examined by dot blot hybridization. The existence of recombinant HBV virion in the culture medium was examined by PCR. Free of packaging signal, HBV genome, which express the HBV structural proteins including core, pol and preS/S proteins, was inserted into pCI-neo vector. HepG2 cell lines were employed to transfect with the construct. G418 selection was done at the concentration of 400mug/ml in the culture medium. The G418-resistant clones with the best expression of HBsAg and HBcAg were theoretically considered as packaging cell lines and propagated under the same conditions. It was transfected with plasmid pMEP-CPAS and then selected with G418 and hygromycin in the culture medium. The existence of recombinant HBV virion in the culture medium was examined by PCR. RESULTS: The mean inhibitory rates of HBsAg were 2.74% 3.83%, 40.08 2.05% (t=35.5, P<0.01), 66.54% 4.45% (t=42.3, P<0.01), and 73.68% 5.07% (t=51.9, P<0.01) in group 2.2.15-pMEP4, 2.2.15-CP, 2.2.15-SAS, and 2.2.15-CPAS, respectively. The mean inhibitory rates of HBeAg were 4.46% 4.25%, 52.86% 1.32% (t=36.2, P<0.01), 26.36% 1.69% (t=22.3, P<0.01), and 59.28% 2.10% (t=39.0, P<0.01), respectively. The inhibitory rates of HBc related HBV DNA were 0, 82.0%, 59.9%, and 96.6%, respectively. Recombinant HB virion was detectable in the culture medium of all the three treatment groups. G418-resistant HBV packaging cell line, which harbored an HBV mutant whose packaging signal had been deleted, was generated. Expression of HBsAg and HBcAg was detectable. Transfected with plasmid pMEP-CPAS, it was found to secrete recombinant HB virion and no wild-type HBV was detectable in the culture medium. CONCLUSIONS: It has stronger anti-HBV effects by combined expression of antisense RNA and dominant negative mutants than by individual expression of them. With the help of wild-type HBV, the modified HBV genome can form and secret HBV like particles, which provides evidence that the antiviral gene will be hepatotropic expression and the antiviral effects will be amplified. The packaging cell line can provide packaging for replication-defective HBV, but with low efficiency.

Cell Line↗

PubMed2002

[Preimplantation genetic diagnosis].

OBJECTIVE: To evaluate the clinical effectiveness of preimplantation genetic diagnosis (PGD) for the couples having a higher risk of genetic diseases with fluorescence in situ hybridization (FISH). METHODS: Ovarian hyperstimulation, oocyte retrieval and in vitro fertilization were performed in 4 cases. Blastomere biopsy were performed from the embryos at the 6 - 8 cell stage. Genetic diagnosis was identified by single blastomere FISH. The female embryos or normal embryos were transferred into uterus. RESULTS: Twelve embryos were available for embryo biopsy. Twenty cells were collected. Seventeen nuclei were found after fixation. Hybridization signals of 11 embryos were clear and 8 embryos were transferred into uterus. One clinical pregnancy was achieved, and 1 normal female infant was delivered on september 14, 2001. CONCLUSION: PGD is very valuable for avoiding selective abortion and birth of affected child of the couples having a higher risk of genetic diseases by detecting embryo sex and chromosome number using FISH.

Adult↗

PubMed2002

[Effect of G-CSF and GM-CSF on expression of TNF-alpha mRNA and CD69 and secretion of IgG in peripheral blood mononuclear cells from systemic lupus erythematosus patients].

In order to understand the effect of hematopoietic stem cell mobilization agents, G-CSF and GM-CSF, on the expression of TNF-alpha mRAN and CD69 and secretion of IgG in SLE patients' peripheral blood mononuclear cells (PBMNC), expression of TNF-alpha mRNA and CD69 was measured by RT-PCR and flow cytometry, respectively, and IgG secretion by ELISA. The results showed that 0.1 - 2.0 microg/ml G-CSF did not affect the expression of TNF-alpha mRNA in active and static patients' PBMNC treated with 0.1 - 2.0 microg/ml cytokines, and 2.0 microg/ml GM-CSF increased the expression of TNF-alpha mRNA in active patients' PBMNC. G-CSF and GM-CSF did not interfere the expression of CD69 in active and static patients' PBMNC, however, the expression of CD69 was significantly increased in active patients' PBMNC treated with GM-CSF at more than 8 microg/ml. There was no obvious change of IgG secretion from PBMNC induced with 10 microg/ml G-CSF, while the IgG secretion was stimulated by 10 microg/ml GM-CSF. It was concluded that G-CSF as a mobilization agent could be safe to SLE patients, but GM-CSF may cause some harmful effects to patients because of the increase of the parameters relating to activity of lupus in active SLE patients.

Antigens, CD↗

PubMed2002

[Comparison of detection of trisomy 8 with fluorescence in situ hybridization and conventional karyotype analysis in myelodysplastic syndrome].

The purpose of this study was to compare the detection of trisomy 8 in myelodysplastic syndrome (MDS) patients with interphase fluorescence in situ hybridization (FISH) and cytogenetic karyotype analysis. Using Spectrum Green labeled chromosome 8 centromere probe, interphase FISH was established. The trisomy 8 clones were simultaneously detected in 48 MDS cases with FISH and conventional cytogenetic analysis (CCA). Results showed that the CCA revealed no significant difference of constitutional proportion between MDS-RA and MDS-RAEB with karyotypes of whole +8, partial +8 and one +8. With FISH, detectable rates were 66.1% for whole +8. Partial +8 and sole +8 were significantly higher than one +8 and complex +8, respectively. The percentages of trisomy 8 were similar in MDS-RA and MDS-RAEB. Trisomy 8 was detected in 1 of 15 specimens with normal or abnormal karyotype without trisomy 8 by FISH. There was linear correlation between the percentages of partial +8 detected by FISH and CCA. Two patients received CCA and FISH examination at diagnosis and during treatment, the percentage of trisomy 8 was increased with progress of disease. In conclusion, our results showed that FISH is a sensitive and accurate technique to detect trisomy 8 in MDS patients. It can provide contribute to diagnosis, assessment of curative effect and predicting progress of disease in MDS. Clone size of trisomy 8 does not related to classification of MDS, but sole +8 is seems to see in MDS-RA frequently.

Chromosomes, Human, Pair 8↗

PubMed2002

[In vitro inhibition and mechanism of multiple myeloma cells growth by thalidomide].

To investigate the influence of the thalidomide on the growth of multiple myeloma cells from untreated, relapsed or refractory patients and summarize its mechanisms, thalidomide influence on colony growth of untreated, relapsed or refractory multiple myeloma cells cultured by semisolid methylcellulose was observed. The level of interleukin-6 (IL-6) autosecreted by myeloma cells was tested by IL-6-dependent cell line when myeloma cells were treated with thalidomide at 200 microgram/ml, and in the same concentration of thalidomide the expression of IL-6 receptor were tested by flow cytometry. Results showed that colony growths of myeloma cell from untreated and relapsed or refractory patients were all colonies were inhibited when treated by thalidomide up to 75 microgram/ml or 100 microgram/ml concentration. The inhibition was concentration-dependent, higher concentration cause more inhibition. After treatment with thalidomide at 200 microgram/ml, the concentrations of IL-6 secreted by myeloma cells were (148.5 +/- 96.7) microgram/ml, and the levels of IL-6 receptor expressed on the cell surface were 16.7% and 20.2% in untreated and relapsed or refractory patients, respectively, and those were significantly lower than those levels in the cells before exposure to thalidomide. It was concluded that thalidomide can inhibit growth of both relapsed or refractory cells and untreated myeloma cells in vitro. Therefore, it can be used to treat untreated multiple myeloma patients. Inhibiting tumor cells secreting level of IL-6 and reducing the expression of IL-6 receptor on myeloma cell surface is one of the mechanisms for thalidomide to remedy multiple myeloma patients

Angiogenesis Inhibitors↗

PubMed2002

[Influence of thalidomide on interleukin-6 and its transmission in multiple myeloma patients].

OBJECTIVE: To evaluate the mechanism and influence of thalidomide on interleukin-6 (IL-6), IL-6 receptor (IL-6R) and its transmitting chain in multiple myeloma patients. METHODS: Serum level of IL-6, expression of IL-6R on myeloma cells and IL-6R beta mRNA in multiple myeloma patients were measured by enzyme linked immunosorbent assay (Elisa), flow cytometry and reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Serum level of IL-6 in multiple myeloma patients was 564.8 +/- 319.4 ng/L, with a positive rate on the myeloma cells of 33.6% before oral 200 mg/d thalidomide. They were 560.3 +/- 414.8 ng/L and 31.8% on D14 after oral 200 mg/d thalidomide, which were not significantly different as compared with those before (P > 0.05). On D14, 28, 42, 56 and 84 after oral 400 mg/d thalidomide, the serum level of IL-6 in multiple myeloma patients were 516.7 +/- 131.9 ng/L, 426.7 +/- 180.4 ng/L, 387.9 +/- 187.4 ng/L, 350.1 +/- 85.5 ng/L and 212.3 +/- 92.5 mg/L, with positive rates on the myeloma cells of 28.5%, 24.3%, 21.3%, 12.6% and 10.1%, which were all lower than those before oral 200 mg/d thalidomide (P < 0.05 or P < 0.01). Ratios before and on D14 after oral 200 mg/d thalidomide were 7.8 and 6.9, with no statistical significance (P > 0.05). Ratios on D14, 28 after oral 400 mg/d thalidomide were 5.3 and 2.7, which were lower than those before oral 200 mg/d thalidomide (P < 0.01). CONCLUSION: Reduction of serum level of IL-6 in multiple myeloma patients and decrease in IL-6R expression on the myeloma cells and IL-6R beta mRNA occur on D14 after oral 400 mg/d thalidomide. These changes become more obvious with time. The antitumor mechanism of thalidomide may be related to reduction of IL-6 serum level in multiple myeloma patients and decrease in IL-6R expression on the myeloma cells and IL-6R beta mRNA.

Aged↗

PubMed2002

[Combination of thalidomide and rituximab in suppressing myeloma cells in vitro].

BACKGROUND & OBJECTIVES: The efficiency of rituximab (Mabthera) is related to CD20 expression density on cell membrane. It is not yet to be solved how to heighten expression level of CD20 on multiple myeloma (MM) cell membrane and to increase the efficacy of Mabthera to MM. This study was designed to observe whether thalidomide could promote the effect of Mabthera on suppressing myeloma cells in vitro and its possible mechanism. METHODS: Colony growth of 18 untreated and 20 relapsed or refractory MM patients' myeloma cells were observed in the methylcellulose semisolid medium adding thalidomide (10, 50, 75, 100, 150, 200, 300 micrograms/ml) or Mabthera (0.5, 1, 2, 4, 8, 12, 16 micrograms/ml) or thalidomide above 7 doses in combination with Mabthera of 16 micrograms/ml or Mabthera above 7 doses in combination with thalidomide of 75 micrograms/ml. Change of CD20 expression on the myeloma cells were measured by flow cytometer after and before myeloma cells were treated with thalidomide. RESULTS: The inhibition of the colony formation of untreated MM patients' myeloma cells occurred in 1. use only of thalidomide at more than or equal to 75 micrograms/ml or use only of Mabthera at 16 micrograms/ml, 2. use of thalidomide at 75 micrograms/ml with or without Mabthera at 16 micrograms/ml, 3. use of thalidomide at more than or equal to 75 micrograms/ml with or without Mabthera at 16 micrograms/ml; The inhibition of the colony formation of relapsed or refractory MM patients' myeloma cells occurred in 1. use of thalidomide at 75 micrograms/ml with Mabthera at 16 micrograms/ml, 2. use of thalidomide at more than or equal to 100 micrograms/ml with or without Mabthera at 16 micrograms/ml; Thalidomide at more than 75 micrograms/ml enhanced the expression of CD20 antigen in untreated and relapsed or refractory MM patients' myeloma cells. CONCLUSION: Thalidomide could enhance the inhibition of Mabthera on colony formation of MM patients' myeloma cells, which is related to that thalidomide enhances CD20 antigen expression of myeloma cells.

Angiogenesis Inhibitors↗

PubMed2000

[In Vitro Expansion of Bone Marrow Stem/Progenitor Cells by Human Placental Cell-Free Suspension]

In order to explore the expansive effect of human placental cell-free suspension (HPCFS) on bone marrow hematopoietic stem/progenitor cells, and to compare the effect of HPFCS with some cytokines and their combination, human marrow CFU-GM, CFU-GEMM and BFU-E were assayed in a semisolid methyl cellulose culture system using HPCFS, IL-3, GM-CSF, and IL-3 + IL-6 + GM-CSF + EPO as colony stimulating factors, respectively. The results showed that HPCFS stimulated the growth of CFU-GM, CFU-GEMM and BFU-E and the optimal concentrations for stimulating effect were 200 - 300 micro g protein/L, and the yield of 3 kinds of colony in HPCFS group was higher than that in IL-3, GM-CSF and IL-3 + IL-6 + GM-CSF + EPO groups. The expansive effect of HPCFS on marrow progenitors was superior to IL-3, GM-CSF and IL-3 + IL-6 + GM-CSF + EPO. Human placental cell-free suspension contained a variety of cytokines to stimulate proliferation of hematopoietic stem/progenitor cells, and it could be used as an efficacious and inexpensive agent to expand hematopoietic stem/progenitor cells in vitro.

Journal Article↗