[Primary culture and identification of rat glomerular podocytes].

OBJECTIVE: To establish an easy and feasible method for primary culture and identification of rat glomerular podocytes. METHODS: Glomeruli from Sprague-Dawley (SD) rats weighing 60-100 gram were isolated by the method of different size combination of screen. Isolated glomeruli were appropriately digested with 2 g/L type IV collagenase and cultured in 25 cm2 plastic flask coated with rat tail collagen in K1-3T3 medium with ITS-X (containing insulin-transferrin-selenium). Subculture of primary cultured epithelial cells was performed at 9-10 days after implantation of collagenase digested glomeruli. Podocytes were identified by the morphology study with scanning electron microscope and inverted microscope, as well as the immunohistochemistry staining (SP methods) study for the expression of keratin, desmin and Wilms' tumor suppressor-1 (WT-1). RESULTS: Epithelial cells outgrowth from isolated glomeruli appeared after 3 days primary culture and grew to confluence with cobblestone-appearance at 9-10 days. These cobblestone cells were subcultured at this point and gradually conversed into large, flat arborized cells with well-developed processes and microvilli. These arborized cells were negative expression with desmin staining and showed positive expression of cytokeratin and WT-1, which indicated that they were podocytes. CONCLUSION: Implantating collagenase digested-glomeruli is an easy and feasible method for primary culture of rat glomerular podocytes. WT-1 may serve as a good marker to identify rat glomerular podocytes.

[Influence of mutation of -1997G-->T of COL I A1 gene on the biochemical function of osteoblast].

OBJECTIVE: To study the influence of mutation of -1997G-->T of COL I A1 gene on the biochemical function of osteoblast, and the pathomechanism of BMD. METHODS: Spongy bones were obtained to culture osteoblast primarily during total hip or knee replacements. The genotype of osteoblast was identified with PCR-RFLP. The amount of mRNA of COL I A1 and collagen type I were determined by RT-PCR and ELISA. The growth of osteoblast, the activity of bone ALP, and the amount of calcium in cell matrix and calcium nodus were measured. RESULTS: Three genotypes GG, GT and TT in osteoblast were successfully identified. Compared with GG and GT genotypes, lower expression of mRNA of COL I A1 gene, lesser collagen type I, calcium in cell matrix, and calcium nodus were found in the cells with TT genotype (P < 0.01). No significant differences were found between GG and GT genotype (P > 0.05). There were no significant differences in age, growth of osteoblast, and activity of bone ALP among the three genotypes (P > 0.05). CONCLUSION: Cells with TT genotype have low expression of mRNA of COL I A1 gene and less collagen type I , calcium in cell matrix and calcium nodus. The lower amount of collagen type I synthesized by osteoblast can decrease the matrix outside the bone cells and result in insufficient site for calcium deposition. This may be the cause of lower BMD in patients with TT genotype.

[Treatment of early avascular necrosis of femoral head by core decompression and transplantation of human hepatic growth factor gene-modified osteoblasts: experiment with rabbits].

OBJECTIVE: To evaluate the effects of transplantation of human hepatocyte growth factor (hHGF) gene-modified osteoblasts combined with core decompression in treatment of avascular necrosis of femoral head (ANFH). METHODS: The plasmid pcDNA3.1(+)-hHGF containing hHGF gene was constructed. Osteoblasts were isolated from fetal rabbits, cultured, and transfect3d with the plasmid pcDNA3.1(+)-hHGF or blank plasmid pcDNA3.1(+), or used as controls. Thirty-six adult New Zealand rabbits were made into ANFH models, underwent core decompression, and were randomly divided into 3 groups. Group A, transplanted with osteoblasts transfected with pcDNA3.1(+)-hHGF plasmid, Group B, transplanted with osteoblasts not transfected with pcDNA3.1(+)-hHGF plasmid, and Group C, injected with PBS medium. 2, 4, and 8 weeks later samples of femoral head were obtained to undergo CT, histological examination, and capillary ink infusion so as to observe the angiogenesis and osteogenesis. RESULTS: The pcDNA3.1(+)-hHGF transfected osteoblasts showed stable expression of hHGF. The numbers of newly formed vessels of the femoral heads of the group transfected with pcDNA3.1(+)-hHGF-transfected osteoblasts 2 and 4 weeks later were (29.47 +/- 1.64) and (34.02 +/- 1.72)/cm2 respectively, both significantly higher than those of the group transfected with blank plasmid-transfected osteoblasts [(20.61 +/- 1.91) and (25.57 + 2.20)/cm2 respectively, both P <0. 01]. Eight weeks later the numbers of mature trabecular bone and bone marrow of Groups A and B were significantly higher than those of Group C. CONCLUSION: Core decompression combined with transplantation of HGF gene-modified osteoblasts promotes angiogenesis, enhances bone formation, and improves the restoration of avascular necrosis of femoral head.

[Construction and expression of recombinant plasmid pcDNA3.1(+) -hHGF available in osteoblast].

OBJECTIVE: To construct a plasmid carrying human hepatocyte growth factor (hHGF) gene and determine the effects of the hepatocyte growth factor (HGF) gene on proliferation and differentiation of osteoblast in vitro. METHODS: The full length cDNA of hHGF, which was amplified from human liver mRNA by RT-PCR, was cloned into pcDNA3.1(+) vector to construct pcDNA3.1(+) -hHGF recombinant plasmid. With lipofectamine, the recombinant plasmid pcDNA3.1(+) -hHGF was used to transfect the culture osteoblasts. After pcDNA3.1(+) - hHGF transfection, the positive cell clones were selected with G418. The stable transfection and expression of hHGF in the osteoblasts were measured by immunohistochemical staining and RT-PCR respectively. The effect of pcDNA3.1(+) -hHGF transfection on osteoblast proliferation was measured by MTT colorimetric assay. Flow cytometer was used to determine the effect of pcDNA3.1(+) -hHGF transfection on cell cycle of osteoblast. The quantitive detection of hHGF expression was performed through ELISA. Alkaline phosphatase (AP) was also detected using enzyme kinetics. RESULTS: The recombinant plasmid pcDNA3. 1(+) -hHGF was identified by restriction endonuclease digestion and nucleotide sequencing. Osteoblasts could be effectively transfected by pcDNA3.1(+) -hHGF with lipofectamine in vitro. The stable expression of hHGF in pcDNA3.1(+) -hHGF transfection osteoblast was confirmed. Human HGF protein was detected by immunohistochemical staining and RT-PCR in osteoblasts 4 weeks after cell clone selected with G418. pcDNA3.1(+) -hHGF gene transfer responsible to improve the proliferation was proved by MTT assay, flow cytometer showed cellular proportion in "S" phase obviously increased. AP activity in transfected cells was increased significantly. CONCLUSIONS: Osteoblasts which express stable and high-level hHGF was established successfully, exogenous hHGF gene could stimulate the proliferation, differentiation and function of osteoblast and could be applied further to gene therapy.

[Inhibitory effects of deoxyribozyme on the expression of Period1 gene in vitro and on morphine-induced psychic dependence in mice].

OBJECTIVE: To investigate the regulatory effects of deoxyribozyme on the expression of Period 1 gene in vitro and on the morphine-induced psychic dependence in mice. METHODS: The specific deoxyribozymes toward Period 1 mRNA was designed by MFold analysis and synethsized chemically. By LipofectAMINE mediated DNA transfection technique, DRz164 and pcDNA3-Per1 were introduced into NIH3T3 cells. The effects of deoxyribozyme on Period 1 gene were studied by reverse transcript-polymerase chain reaction (RT-PCR) and flow cytometry(FCM). The morphine-induced reward in mice was observed in a conditioned place preference test after pretreatment of the mice with the intracerebroventricular administration of deoxyribozyme. RESULTS: After NIH3T3 cells were transfected by pcDNA3-Per1 and DRz164, the Period 1 mRNA was reduced by 42.4%. And PERIOD proteins were decreased by 57.5%. After being pretreated with deoxyribozyme, the mice did not show morphine-induced place preference. CONCLUSION: DRz164 can highly block the expression of Period 1 gene, which cleaves the Period 1 mRNA in the transfected cells specifically. The suppression of morphine-induced place preference can be effected by pretreating the mice with alleviating their psychic dependence on morphine.

[Inhibitory effect of human brain myelin basic protein on H2O2-induced apoptosis of human lung cancer cell line YTLMC-90].

BACKGROUND & OBJECTIVE: Human brain myelin basic protein (MBP) distributes in nervous system and other tissues extensively, and can be detected in many kinds of tumor cells, such as lung cancer, breast cancer, and neuroglioma. However, it has not been reported whether MBP is relevant to the activity of neural invasion of tumors and whether MBP plays a role in biological behaviors of human lung cancer cells. This study was to investigate the inhibitory effect of MBP on hydrogen peroxide (H2O2)-induced apoptosis of human lung cancer cell line YTLMC-90. METHODS: YTLMC-90 cells were transfected with plasmid pSVCEPMBPCAT containing MBP cDNA minigene (test group), or empty vector pSVCEPCAT, or received no transfection (control group), and exposed to H2O2. The expression of MBP in YTLMC-90 cells was detected by Western blot. Cell proliferation was measured by MTT assay. The morphologic and ultra-structural changes of apoptotic cells were observed by microscopy with fluorescent staining of acridine orange (AO) and electron microscopy. The DNA fragmentation was examined by agarose gel electrophoresis. RESULTS: After exposed to 200 micromol/L H2O2 for 24 h, the inhibitory rate of cell growth was significantly lower in test group than in empty vector group and control group (36.67% vs. 78.67% and 84.00%, P<0.001). The morphologic and biochemical changes of apoptotic cells, such as shrinkage of cytoplasm and nucleus, fragmentation of chromatin, and ladder pattern of DNA, were commonly observed in cells in control group, but these apoptotic features were not discovered in test group. CONCLUSION: MBP markedly inhibits H2O2 cytotoxicity to YTLMC-90 cells through promoting cell proliferation and antagonizing H2O2-induced apoptosis.

[Feasibility of cationic lipid mediated nm23-h1 plasmid transfecting adnoid cystic carcinoma in vivo].

OBJECTIVE: To investigate the feasibility of plasmid nm23-h1 transfection on high metastatic potential adnoid cystic carcinoma (ACC-M) cell line mediated by cationic lipid. METHODS: ACC-M cell were implanted in the maxillofacial region in each of 40 BALB/c nude mice. After the tumor growth to 1 cm in diameter, 0.1 ml Lipofctamine-nm23-hl plasmid complex were injected intratumorally in 10 mice, 3 days after the first injection, 10 mices injected for twice, 10 mice as plamid-blank control, another 10 mice were injected 0.2 ml complex, 2, 3, 7days after the injection, the mice were killed and the specimen for HE and immunohistological chemistry study. RESULTS: nm23-h1 expression initiated in the tumor cells 3 days after the complex injection, 7 days later, the expression level increased accompanying with extracellular matrix increase, twice injection and multiple channel injection would gain better nm23-h1 expression than once injection and single-channel injection respectively. CONCLUSION: Cationic lipid mediated nm23-h1 plamid transfecting adnoid cystic carcinoma can gain small range positive expression, but the results give little prospect for further clinical treatment in such a manner.

[Impact of p16INK4A and p15INK4B on human hepatocellular carcinoma cell proliferation and apoptosis].

OBJECTIVE: Both tumor suppressor p16INK4A and p15INK4B are members of INK family of CDK inhibitor. Although the role of p16 has been well documented, the role of p15 and its signaling pathway remain less well studied. This study was aimed to assess the effect of p16 and p15 on hepatocarcinoma cell lines with different status of Rb gene. METHODS: After identification of the genetic status of p16, p15 as well as Rb of human hepatocellular carcinoma (HCC) cell lines BEL7402, SMMC7721 with the use of multiple PCR, the eukaryotic expression p16 and p15 recombinants pXJ-p16 and pXJ-p15 were constructed, respectively. The existence of exogenous p16, p15 genes, and the expression of p16 and p15 were assayed by means of PCR and RNA dot blotting. Finally, the proliferation and apoptosis were studied by using MTT, colony formation assay and flow cytometry. RESULTS: Neither deletion of p16 nor p15 was detected in the two cell lines. However, Rb exons 14-16 instead of exons 22-23 deletion existed in SMMC7721. The increased mRNA expression level of p16 was found in BEL7402-p16 and SMMC7721-p16, while increased mRNA expression level of p15 was found in BEL7402-p15. The endogenous p16 and p15 genes were transcripted at low level. The cell growth and colony formation were decreased in BEL7402-p15, compared with either mock cell BEL7402 or vector control cell BEL7402-pXJ. Also shown in this study were an altered G1 phase population from 37.7% to 43.6%, an S phase population from 22% to 13% (P<0.05), and a Sub G1 peak (apoptosis peak) in BEL7402-p15. Conversely, BEL7402-p16 with endogenous p16 gene showed neither difference in cell cycle population nor difference in colony formation rate, compared with control cell groups. Additionally, SMMC7721-p16 cell growth was not inhibited by exogenous p16 gene. CONCLUSION: p15 significantly arrested cell proliferation and induced apoptosis in BEL7402 in vitro, and the function was not influenced by endogenous p15 gene. The inhibition of cell growth by p16 on HCC cells could be dependent on intact RB pathway.

[Suppressive effect of brain derived neurotrophic factor on H2O2-induced apoptosis in human lung cancer cell YTMLC-90].

BACKGROUND & OBJECTIVE: It has been shown that neurotrophic factors such as nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), and neurotrophin (NT-3/4) are synthesized in a variety of cells inside and outside the nervous system. These factors are not only able to promote neural survival, proliferation and apoptosis of neural cells but also relevant to the activity of neural invasion of tumors. It has not been reported to date whether BDNF may play roles in the biological behavior of human lung cancer cells. The aim of this experiment was to investigate the effect of BDNF on hydrogen peroxide (H2O2)-induced apoptosis in the human lung cancer cell line YTMLC-90. METHODS: The minigene pSVCEPBFCAT containing the promoter and enhancer elements of the human a1(I) collagen gene(COLIA1) at its 3' terminus followed by hBDNF gene cDNA was transfected and derived BDNF ectopic expression in the human lung cancer cells. The cell proliferation was measured by MTT assay. The morphological and ultra-structural changes of apoptotic cells were observed by microscopy with fluorescent stain of acridine orange and electron microscopy. The DNA fragmentation was examined by agarose gel electrophoresis. RESULTS: After exposure of growing cells to 200 micromol/L H2O2 for 24 hours, the inhibition rate of cell growth was 30% in the pSVCEPBFCAT-transfected YTMLC-90, 84.60% in controls of non-transfected YTMLC-90, and 80.00% in pSVCEPCAT-transfected YTMLC-90, respectively (P< 0.001). The morphological and biochemical changes of apoptotic cells such as shrinkage of cytoplasm and nucleus,fragmentation of the chromatin, and ladder pattern of DNA were commonly observed in the cell population of controls, but these apoptotic features were not discovered in the pSVCEPBFCAT-transfected YTMLC-90. CONCLUSION: BDNF markedly inhibits H2O2 cytotoxicity on human lung cancer cell YTMLC-90 by promoting YTMLC-90 proliferation and antagonizing H2O2-induced apoptosis.

[Encapsulated ANP cDNA transfection cells attenuate hypertension in hypertensive rats].

OBJECTIVE: We Investigated a gene therapy delivery system based on microcapsules enclosing recombinant Chinese hamster ovary (CHO) cells engineered to secrete a therapeutic peptide-atrial natriuretic peptide (ANP). METHOD: Human atrial natriuretic peptide gene transfecting Chinese hamster ovary (CHO) cells were encapsulated in non-antigenic biocompatible polycaprolactone (PCL) capsules prior to their implantation into rats, then, the PCL-tubes were implanted into hypertensive DSS rats intraperitoneally. RESULT: The PCL-tubes 2 d post implantation caused a significant delay of blood pressure increase. The effect lasted for more than 5 months. The PCL-tubes also caused significant increases in renal blood flow, glomerular filtration rate, sodium output, urine excretion. Plasma levels of ANP in rats implanted with the PCL-tubes containing engineering cells is higher than that of the control rats. CONCLUSION: This study demonstrates encapsulated engineering cells have significant potential in treatment of hypertension.