ObjectiveTo review the characteristics of human defensin 5 (HD-5), including molecular structure, antibacterial activity and gene expression, and to show its development prospect as a new drug in the treatment of enterogenic infection. MethodsThe published papers about HD-5 were reviewed to summarize its research progress. ResultsBeing a 3-5 KDa cationic peptide rich in cysteine and arginine, especially without glycosylated side-chain, HD-5 plays its antibacterial role against positive and negative Gram’s bacterium, fungi, spirochete, protozoan and enveloped virus with the special active center composed of three disulfide bonds. HD-5 encoding gene alpha defensin 5 (DEFA5) localizes at the 8th chromosome P21-pter with 449 bp, which includes five pieces of sequence: 5′ untranslated region (1-40)，signal peptide (41-97), propiece (98-226), mature peptide (227-322)，and Poly A (433-438).ConclusionAs a broad-spectrum and effective endogenous antimicrobial peptide, HD-5 would be a promising alternative peptide against enterogenic infection if the accessibility to its mass production is settled.
Objective To investigate the effect of human tooth bone graft materials on the proliferation, differentiation, and morphology of macrophages, and to understand the biocompatibility and cytotoxicity of human tooth bone graft materials. Methods Fresh human teeth were collected to prepare human tooth bone graft materials, the adhesion of mouse mononuclear macrophages RAW264.7 to human bone graft materials was observed under confocal microscopy. Scanning electron microscopy was used to observe the morphology of human tooth bone graft materials, OSTEONⅡ synthetic highly resorbable bone grafting materials, and untreated tooth powder (dental particles without preparation reagents). Different components of the extract were prepared in 4 groups: group A (DMEM medium containing 10% fetal bovine serum), group B (human tooth bone graft materials), group C (OSTEONⅡ synthetic highly resorbable bone grafting materials), group D (untreated tooth powder without preparation reagents). The 4 groups of extracts were co-cultured with the cells, and the cytotoxicity was qualitatively determined by observing the cell morphological changes by inverted microscope. The cell proliferation and differentiation results and cell relative proliferation rate were determined by MTT method to quantitatively determine cytotoxicity. The cell viability was detected by trypanosoma blue staining, and tumor necrosis factor α (TNF-α ) and interleukin 6 (IL-6) expressions were detected by ELISA. Results Scanning electron microscopy showed that the surface of the human tooth bone graft material and the OSTEONⅡ synthetic highly resorbable bone grafting materials had a uniform pore structure, while the untreated tooth particle collagen fiber structure and the demineralized dentin layer collapsed without specific structure. Confocal microscopy showed that the cells grew well on human tooth bone graft materials. After co-culture with the extract, the morphology and quantity of cells in groups A, B, and C were normal, and the toxic reaction grades were all grade 0, while group D was grade 3 reaction. MTT test showed that the cytotoxicity of groups B and C was grade 0 or 1 at each time point, indicating that the materials were qualified. The cytotoxicity was grade 2 in group D at 1 day after culture, and was grade 4 at 3, 5, and 7 days. Combined with cell morphology analysis, the materials were unqualified. The trypanosoma blue staining showed that the number of cells in groups A, B, and C was significantly higher than that in group D at each time point (P<0.05), but no significant difference was found among groups A, B, and C (P<0.05). ELISA test showed that the levels of TNF-α and IL-6 in groups A, B, and C were significantly lower than those in group D (P<0.05), but no significant difference was found among groups A, B, and C (P<0.05). Conclusion The human tooth bone graft materials is co-cultured with mice mononuclear macrophages without cytotoxicity. The extract has no significant effect on cell proliferation and differentiation, does not increase the expression of inflammatory factors, has good biocompatibility, and is expected to be used for clinical bone defect repair.
ObjectiveExtracting the endothelial cells or all endothelial cells and interstitial cells from the cryopreserved homograft valves (HV), to evaluate the immunogenicity of this two kinds of decellular HV. MethodsFor extracting the endothelial cells, the leaflet and wall of the HV were decellularized by a 4-step detergent-enzymatic extraction method involving the 1% triton in combination with RNase (1μg/ml) and DNase (10μg/ml). For extracting the endothelial cells and interstitial cells, the leaflet and wall of the HV were decellularized by a 3-step detergent-enzymatic extraction method involving the 1% deoxycholic acid (DOA) in combination with RNase (20μg/ml) and DNase (200μg/ml). HLA-DR antigen expression was detected by using immunohistochemical techniques. The valve and wall of the HV were transplanted subcutaneously in the mice for 8 weeks, and the histology, calcium assay and calcium content were examined. ResultsFor the staining of the HLA-DR antigens, the immunogenic potential of the HV with extracting all endothelial cells and interstitial cells or only the endothelial cells was lower than cryopreserved HV, but it more obviously decreased for the HV with extracting all endothelial cells and interstitial cells. After 8 weeks embedded in the mice, the histological signs of the inflammatory reactions and the calcification extent to the cryopreserved HV and the HV with only extracting endothelial cells were stronger than the HV with extracting all endothelial cells and interstitial cells predominantly. And calcification extent or the inflammatory reactions to the wall of the HV were more severe than those of the leaflet. ConclusionsThe immunogenicity of the HV with extracting all endothelial cells and interstitial cells is much less than HV with only extracting endothelial cells. The histological signs of the inflammatory reactions and the calcification extent in vivo experiments is obviously decreased. For the HV with only extracting endothelial cells, though the histological signs of the inflammatory reactions slightly decrease, the calcification extent in vivo experiments is more severe, especially for the wall. The interstitial cells may be the important factor for the donor-reactive immune responses that is related to the graft calcification or destruction after implantation.
Acquired Immunodeficiency Syndrome (AIDS) is a severe infectious disease induced by human immuno deficiency virus (HIV). Laboratory testing plays an important role in the diagnosis of HIV/AIDS. In general, laboratory testing includes detection of virus antibodies and antigens, virus RNA, immune cells (CD4+, CD8+) and anti-HIV drug resistance. During the past twenty years, great progress has been made in laboratory testing. As research on HIV/AIDS has advanced and biotechnology has developed rapidly, different methods of testing have been discovered. In recent years, the application of molecular biotechnology and immunology has led to important advances for epidemiological surveys, clinical diagnosis and treatment of HIV. The existence of a testing method with high sensitivity and specificity is not only helpful for early diagnosis and prediction, monitoring and evaluation of therapeutic efficacy, but can also reduce the risk of false-negative results. HIV laboratory testing is now developing towards a simple, rapid, sensitive, accurate and automatic way of diagnosing this condition.
Objective To investigate the reversal effect of antisense phosphorothioate oligonucleotide （ASOND) on human hepatoma resistant cells. Methods Human hepatoma resistant cells SMMC-7721 was transfected with synthetic antisense phosphorothioate oligonucleotide complementary to the 5′ region flanking the AUG initiation codon mediated by lipofectamine. In vitro drug sensitivity was measured by MTT assay. The expression of P-170 was determined by flow cytometry and mRNA was assessed by RT-PCR. Results ASOND inhibited the expression of mRNA and p-170 in SMMC-7721, enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug. The best inhibitory effect was achived by the dose of 0.5μmol/L. Conclusion ASOND enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug and reversed the multidrug resistance of SMMC-7721 partially.
Following the peritendon was removed by means of microsurgical technique, the tenocyte was isolated from the human embryonic tendons by digesting it with trypsin and collagenase. These cells were all stored in frozen condition until they were cultured by F12 culture fluid added with 20% FBS to the 15th generation.These cells were able to grow adhering to the wall and stop growing with contact inhibition. The time of cellsgroup duplication was 4 days, which was similar to the peak time of its mitosis. The number of its chromosome group 2n=46 was 87.5-91.0%. The optimal conditions for tendon cell culture in vitro were investigated, and it was found that after they were reaminated and subcultured the frozen storage didn’t influence their growth, morphology, genetic characteristics. In our research we detected the content generation cells and found the cultured human embryonic tenocyte had same ability never changed with the cells subcultured. We also disscussed the future of tenocyte-a biomaterial in the field of artificial implant.
Objective To explore effects of zinc on the contents of cycl in D2, cycl in-dependent kinase 4 (CDK4), and their DNA and total cellular protein in human umbil ical cord blood-drived mesenchymal stem cells (hUCBMSCs). Methods hUCBMSCs were isolated and cultured by density gradient centrifugation adherence method in vitro. At the serial subcultivation, the hUCBMSCs were randomly divided into 7 groups. In control group, hUCBMSCs were cultured with DMEM medium (containing 15%FBS). In treatment groups, hUCBMSCs were cultured with DMEM medium (containing 15%FBS plusZnSO4?7H2O). The final concentrations of zinc were 0.5, 1.5, 2.5, 3.5, 4.5, and 5.5 mg/L, respectively. The cellular surface antigens of CD29, CD34, CD44, and CD45 at the 3rd generation of hUCBMSCs were detected by flow cytometry. MTT assay was used to detect cell activity of the 3rd generation of hUCBMSCs. The optimum concentration of zinc was selected by the results of MTT as experimental group. The cell growth curves of experimental group and control group were drown by counting cell. The cell surface antigen, reproductive cycle, and DNA content were detected by flow cytometry motheds. The contents of cycl in D2 and CDK4 were detected by Western blot method. Results The positive expression rates of CD29 and CD44 were more than 70% in hUCBMSCs. The cell activity of 2.5 mg/L treatment group was superior to other treatment groups, as experimental group. At 7, 14, and 28 days, the contents of DNA, total cellular protein, cycl in D2, and CDK4 of hUCBMSCs were significantly higher in experimental group than those in control group (P lt; 0.01). The percentage of hUCBMSCs at S stage and prol iferation index in experimental group were also significantly higher than those in control group (P lt; 0.01). Conclusion Zinc (0.5-4.5 mg/L) has the promoting effect on the hUCBMSCs activity, and 2.5 mg/L is the optimal concentration. Zinc (2.5 mg/L) can accelerate the prol iferation and DNA reproduction of hUCBMSCs and increase the contents of cycl in D2 , CDK4, and cellular total protein.
ObjectiveTo summarize the biological characteristics of human epidermal growth factor receptor 2 (HER-2/neu) gene, the expression and meaning of HER-2/neu gene in gastric cancer, and clinical application of targeted medicine of HER-2/neu gene in gastric cancer. MethodsRelated literatures about HER-2/neu gene and gastric cancer were retrieved for a review. ResultsHER-2/neu gene encoded human epidermal growth factor receptor, and it participated in the gene regulation of tumor cell proliferation, invasion, and metastasis through the downstream signal transduction pathway. Amplification of HER-2/neu gene or overexpression of HER-2 was closely bound up to the occurrence and development of gastric cancer, however, whether it could be used as independent prognostic factors of gastric cancer remained to be controversial. Several targeted medicine of HER-2/neu gene had applied to clinical at present, and all of them obtained good short-term effect. ConclusionHER-2/neu gene is a reliable target of gastric cancer and targeted medicine of HER-2/neu gene has a promising prospect.
Objective To verify the potential of the recombinant adeno-associated virus 2 (rAAV2) vector as a strategy for human transforming growth factor β1 (hTGF-β1) gene transfer in degenerative intervertebral discs of rabbit, to investigate the gene transduction efficacy and to quantify the biologic effects on the proteoglycan level after gene transferring. Methods Rabbit models of disc degeneration were established by injecting the 25 μL fibronectin fragment (Fn-f, 1 mmol/ L), 4 weeks later,saline with or without virus was injected directly into 96 lumbar discs of 24 mature New Zealand white rabbits (male or female and weighing 1.7-2.2 kg) which were divided into 3 groups (n=8). Group A received the 25 μL rAAV2-hTGF-β1 (1 × 1012 vg/mL); group B received rAAV2-enhanced green fluorescent protein (rAAV2-EGFP); and group C received PBS. Two rabbits of groups A, C were killed 1 week after injection, the immunohistochemical staining for hTGF-β1 was performed on the sl ices of nucleus pulposus (NP) tissues. At 4, 8, and 12 weeks after gene transferring, NP tissues were harvested and cultured to quantify the changes of the proteoglycan level using 35S-sulfate incorporation assay. The expression of EGFP in group B was observed 12 weeks after injection. Results Immunohistochemical staining showed that extensive and intense positive immunohisochemical staining for hTGF-β1 were seen in group A when compared with group C 1 week after gene transferring. The nucleus pulposus tissues from the group A exhibited an increased synthesis of proteoglycan, which was significantly more than that from groups B and C (P lt; 0.05), and no significant difference was observed between group B and group C. The expression of EGFP in group B was high at 12 weeks. Conclusion The discs injected with rAAV2-hTGF-β1 can highly expressed the therapeutic proteins for more than 12 weeks, it is suggested that rAAV2 should be an valid vector for transferring exogenous genes in the degenerative disc. The therapeutic factors hTGF-β1 can efficiently increase the proteoglycan synthesis of the degenerative NP cells.
OBJECTIVE: To observe the proliferation and differentiation properties of primary human embryonic skeletal myoblasts cultured in vitro. METHODS: The skeletal muscle samples were obtained from 20 to 25-week abortion fetus, the family history of inherited myopathies of parental generation was negative. With a modified method of Blau, the muscle sample was digested with trypsin and collagenase. The isolated cell suspension was a mixture of myoblasts and fibroblasts, the latter was removed by repeated attachment to culture dishes. The morphological, immunohistochemical observation, the proliferation and differentiation of primary myoblasts were studied. RESULTS: The isolated myoblasts were spherical in cell suspension and spindle-like after attached to culture dishes. The myosin specialized immunohistochemical staining was bly positive. A large quantity of skeletal muscle specialized creatine kinase (CK-MM) was synthesized in cultured myoblasts. Additionally, while the cell density of myoblasts increased, the monocyte myoblasts would fused to form multinucleated myotube. All those indicated that the cultured cells were myoblasts. Primary myoblasts proliferated quickly, the doubling time, measured in growth curve, was 4.8 days. CONCLUSION: A large number of myoblasts can be available with digestion and repeated attachment method. The cultured cells can be proved as myoblasts by morphological and immunohistochemical detection. The cultured myoblasts have good ability of proliferation and differentiation.