[Construction of recombinant shuttle plasmid pIMP1-eHER2/neu and screening and identification of its stable Clostridium sporogenes transformants].

AIM: To construct recombinant clostridium sporogenes modified with the extracellular domain of human oncogene HER2/neu, to lay a foundation for further study of its antitumor effect. METHODS: The extracellular domain (ECD) of HER2/neu gene was attached to the downstream of promoter and signal sequence of clostridia endo-1, 4-glucanase (eglAp) by SOE-PCR to construct fusion gene eglAp-HER2/neu, which was then inserted into E.coli-clostridia shuttle plasmid pIMP1 to construct recombinant plasmid pIMP1-eHER2/neu. The recombinant plasmid was firstly transformed into E.coli DH5α.Then the correct construct was identified and introduced into C. sporogenes by electroporation. Positive clones were selected by erythromycin resistance, bacteria PCR were used for verification. RESULTS: Restriction map and sequencing result showed that the sequence and ORF of fusion gene eglAp-HER2/neu in recombinant plasmid pIMP1-eHER2/neu was correct. Bacteria PCR results indicated that the recombinant plasmid pIMP1-eHER2/neu was successfully transformed into C.sporogenes. After more than 20 passages under antibiotic pressure, C.sporogenes transformants could stably carry the recombinant plasmid pIMP1-eHER2/neu. CONCLUSION: Stable C.sporogenes transformants with the recombinant plasmid pIMP1-eHER2/neu are successfully acquired, which laid a foundation for further anti-tumor study.

[The expression and anti-apoptotic function of HCMV IE2 protein controlled by Tet-On system].

During the infection of host cells, IE2 protein is one of the first and most abundantly expressed products of HCMV genome, which plays an important role in the controlling of cell cycle and apoptosis. But the correlation between expression level and anti-apoptotic activity of IE2 protein is still not clear. In this study, we successfully established a HCMV IE2 protein expression cell line that was controlled by Tet-On system. The effect of IE2 protein on cell apoptosis and the expression of p53 was detected under different condition of induction. Our results showed that the IE2 protein could inhibit cell apoptosis induced by TNF-alpha. Additionally, the anti-apoptotic activity of IE2 protein seemed to be relevant to its expression level. However, we failed to detect any difference of p53 expression between the IE2 protein expression and non-expression cells. These data indicated that the IE2 protein might inhibit cell apoptosis through regulating different signal pathways.

[Human cytomegalovirus inhibits the differentiation of human hippocampus neural stem cells].

The objective of present study is to investigate the effect of human cytomegalovirus (HCMV) infection on human hippocampus neural stem cells NSCs differentiation in vitro, Fetal hippocampus tissue was dissociated mechanically and then cultured in proliferation medium with EGF and bFGF. Immunofluorescence method was used to detect the expression of NSCs marker-Nestin within these cells. Cultured in 10% FBS, NSCs began to differentiate. On the onset of the differentiation, HCMV AD169 (MOI=5) was added into the differentiation medium. After 7 days differentiation, the effect of HCMV infection on NSCs differentiation was observed by detecting the rate of nestin, GFAP and HCMV immediate-early (IE) positive cells with confocal microscopy and immunofluorescence method. The resucts showed most of the cells (passage 4-6 ) were Nestin positive and could differentiate into NSE-positive neurons and GFAP-positive astrocytes. On day 7 postinfection, 86% +/- 12% of infected cells were IE positive. The percentage of Nestin-positive cells was 50% +/- 19% and 93% +/- 10% (t= 6.03, P<0.01)and those of GFAP-positive cells was 81% +/- 11% and 55 +/- 17% (t=3.77, P<0.01) in uninfected and infected cells respectively. These findings indicated that NSCs were HCMV permissive cell and HCMV AD 169 infection suppressed the differentiation of Hippocampus-genetic human neural stem cells into astrocytes.

[Detection of tumor necrosis factor mRNA expression in human cervix and endometrium by in situ hybridization].

OBJECTIVE: To investigate the expression and distributions of tumor necrosis factor (TNF) mRNA in cells derived from human cervix and endometrium with different pathological changes. METHODS: Non-isotopic in situ hybridization was used to study the locus expression of TNF mRNA in 3 cell lines (SiHa, W12 and NCx) derived from human cervix and 96 paraffin-embedded cervical specimens along with that in 75 specimens of emdometrial tissues. RESULTS: TNF mRNA expression was detected in SiHa and W12 cell lines but not in NCx cells. The cervical specimens under examination presented TNF mRNA expression in the cells from the epithelium, interstitial tissues, vascular walls and smooth muscles. The mononuclear cells and smooth muscle cells of endometrial carcinoma tissues, but not those of the normal endometrial tissues, were positive for TNF mRNA expression. CONCLUSION: Epithelial cells derived from condylomatosis and squamous cell carcinoma of the cervix are one type of TNF-producing cells, and the intensity of TNF mRNA expression is related to the degree of uterine hyperplasia. Human papilloma virus infection may enhance TNF mRNA expression in the cervical cells.