Biomedical subjects
Publications and source records attributed to Feng Mu.
Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) was isolated from a pig during a survey for possible routes of viral transmission after a SARS epidemic. Sequence and epidemiology analyses suggested that the pig was infected by a SARS-CoV of human origin.
OBJECTIVE: To develop a rapid and efficient method for preparing recombinant adenovirus containing human mIkappaBalpha gene by homogenous recombination in E.coli. and detect its expression in Hep G2 cells. METHODS: The mIkappaBalpha gene was cloned into the shuttle plasmid pAdTrack-CMV containing green fluorescent protein (GFP) reporter gene, followed by linearization of the resultant plasmid pAdTrack-CMV- mIkappaBalpha by Pme I digestion and subsequent cotransformation into E.coli BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1. The recombinant plasmid pAd- mIkappaBalpha was selected for kanamycin resistance and confirmed by multiple restriction endonuclease analyses. Finally, the linearized recombinant plasmid was transfected into 293 cells, in which Ad- mIkappaBalpha were generated within 7 to 10 days. The virus titer in 293 cells and its infection efficiency in Hep G2 cells were detected and calculated with the aid of GFP expression. RESULTS: PCR indicated that the recombinant adenovirus contained mIkappaBalpha gene and the titer of Ad- mIkappaBalpha was 2.7x10(9) PFU/ml. With a multiplicity of infection (MOI) of 10, Ad- mIkappaBalpha could be expressed stably and efficiently in Hep G2 cells and the infection efficiency was 57% at 24 h and 100% at 48 h after transfection. CONCLUSIONS: Homogenous recombination in E.coli can efficiently and conveniently construct recombinant adenovirus containing mIkappaBalpha gene capable of amplification in 293 cells and efficient infection of Hep G2 cells. The recombinant adenovirus may serve as a good gene transfer vector for study the function of mIkappaBalpha gene and therapy for hepatocarcinoma.
AIM: SARS-associated coronavirus (SARS-CoV) has been confirmed as the pathogen for severe acute respiratory syndrome (SARS). The aim of our study was to construct a sensitive and specific real-time quantitative fluorescent (QF) reverse transcriptase (RT)-PCR method for the detection of SARS-CoV RNA. METHODS: Stored blood specimens from 44 patients with confirmed SARS were used along with blood samples from two sets of controls, 30 healthy volunteers who had no contact with SARS patients, and 30 healthy doctors and nurses who had contact with SARS patients but were without symptoms of SARS. Two pairs of primers were synthesized by the Shanghai Sangon Company according to SARS-CoV BJ01 strain sequence (AY278488), and then a pair of primers were designed and compared with a pair of primers published by WHO. RESULTS: Using serial dilutions of SARS-CoV, the 44 blood samples from SARS patients specimens were tested. Using a 0.01% dilution of SARS-CoV, all 44 clinical samples tested positive in our assay. In comparison, using a 0.1% dilution of SARS-CoV, 26 of the 44 samples tested positive using the WHO primers. In the QF-RT-PCR assay, there was a linear amplification from 100 copies to 10(8) copies of the control RNA per RT-PCR and at least 10 copies, and sometimes even 1 copy, of target RNA tested positive in our assay. CONCLUSION: The primer we developed is sufficiently sensitive and specific to diagnose symptomatic SARS-CoV infections and for monitoring virus load.
To understand the time-course of viraemia and antibody responses to severe acute respiratory syndrome-associated coronavirus (SARS-CoV), RT-PCR and ELISA were used to assay 376 blood samples from 135 SARS patients at various stages of the illness, including samples from patients who were in their early convalescent phase. The results showed that IgM antibodies decreased and became undetectable 11 weeks into the recovery phase. IgG antibodies, however, remained detectable for a period beyond 11 weeks and were found in 100 % of patients in the early convalescent phase. SARS-CoV viraemia mainly appeared 1 week after the onset of illness and then decreased over a period of 1 month, becoming undetectable in the blood samples of the convalescent patients. At the peak of viraemia, viral RNA was detectable in 75 % of blood samples from patients who were clinically diagnosed with SARS 1 or 2 weeks before the test.
OBJECTIVE: To observe the expression of mIkappaBalpha, the inhibitor of NFkappaB, in HepG2 heptocellular carcinoma cells and the inhibitory effect of mIkappaBalpha on HepG2 cell growth. METHODS: The adenovirus containing mIkappaBalpha or the packaging vector were harvested from 293 packaging cells to infect HepG2 cells, in which the expression of green fluorescence protein (GFP) and mIkappaBalpha protein were detected. The plating efficiency and colony-forming ability in soft agar were evaluated, cell growth curve was generated and the tumor growth observed in nude mice. RESULTS: HepG2 cells infected by the packaged adenovirus (HepG2/Adv cells) had positive GFP expression, and those infected by the adenovirus containing mIkappaBalpha (HepG2/Ad-mIkappaBalpha cells) showed mIkappaBalpha protein expression, as demonstrated by Western blot analysis. HepG2/Adv cells exhibited markedly lowered colony-forming ability as compared with that of the HepG2/Adv and HepG2 cells, and the latter two cells, but not the former cells, could survive in soft agar and form colonies in the shape of mulberries. Cell growth curve showed that the Hep G2/Ad-mIkappaBalpha cells had also significantly reduced growth rate, but their saturation density in culture was comparable with the other two cells. Four weeks after inoculation in nude mice, HepG2/Ad-mIkappaBalpha and HepG2/Adv cells showed no significant difference in tumorigenicity (80% vs 100%, respectively), but the tumor volume generated by the former cells was significantly smaller (P<0.05). CONCLUSION: mIkappaBalpha gene can inhibit the growth of HepG2 heptocellular carcinoma cells.
OBJECTIVE: To evaluate the diagnosis and treatment of tracheobronchial ruptures due to thoracic trauma. METHODS: The clinical data of 17 cases of bronchial rupture caused by chest trauma was analyzed retrospectively. The surgical approaches and post-trauma complications were described. RESULTS: End-to-end anastomosis was performed in 14 cases. Among the other 3 patients, one received lobectomy after bronchial repair, another had tracheal repair, and resection of the stenosed segment along with end-to-end anastomosis during thoracotomy was necessitated in the last case. Anastomotic stricture occurred early after the operation in 2 of all these cases, while 2 months postoperatively, all the patients were free from such strictures. CONCLUSION: Early diagnosis and operation in such cases can be life-saving, and may warrant early recovery of the pulmonary function. Accurate diagnoses of bronchial rupture can be obtained by bronchoscopy, with which the surgeons can easily locate the rupture during surgery.
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A right single lung transplantation was performed in a 17-year-old female patient with end-stage cystoid pulmonary fibrosis and without cardiopulmonary bypass in June 2003. The donor lung was perfused with cold UW solution with a cold ischemic time of 280 min. The patient weaned from ventilator on the next day of operation (18 h later) and was able to walk at the fourth day postoperatively. Immunosuppression included methylprednisolone used before FK 506, mycophenolate mofetil and prednisone dosed after operation. The patient remains well a month after operation with significant improvement of the lung function and enjoys normal life.
OBJECTIVE: To investigate the structural changes in canine lungs perfused with modified Euro-Collins solution (mECS), thus providing insight into the preservation of the donor lung tissue for transplantation. METHODS: Six dogs were anesthetized and pulmonary perfusion with mECS (4 degrees Celsius) was performed before the lungs were isolated thereafter and stored at 4 to 8 degrees Celsius for 30, 60, 120, 180, 240 min respectively, after which tissue samples of the donor lung were taken for morphological observation with both light and transmission electron microscope. RESULTS: Immediately after reperfusion, the pulmonary tissues exhibited clear and intact structures,showing that mitochondrion swelled slightly in the alveolar epithelial cells (AECs). Mild edema occurred in the alveolar wall and the tissues around the veins at 30 min after preservation, which exacerbated to dilapidation of the alveolar wall and obvious tumefaction of the mitochondrion in the AECs at 60 min. At 180 min, rupture of alveolar wall and emergence of the alveolus was observed at 120 min, and a few pulmonary bullae were formed, the AECs (I) of which presented vacuolar changes in the cytoplasma and mitochondrion, with disappearance of the tiny villus of the AECs (II) at 240 min. CONCLUSION: Within 4 h of preservation in mECS, the pulmonary tissues do not undergo obvious changes as signs of injury, which, however, may not be the case after longer preservation.