Objective To study the effects of the periosteum，synovium andcartilage tissues on the gene expressions of proteoglycan, collagen Ⅱ, andnuclear factor kappa B (NF-κB) and to investigate the different effects of these tissues on cartilage regeneration. Methods In 20 New Zealand white rabbits, 20 cartilage explants were taken from the knee joints in each rabbit, the sizeof which was 4 mm×4 mm×4 mm. All the cartilages were divided into the following 4 groups and cultured for 7 days: Group A, with 5 pieces (2 mm×2 mm) of the synovium of theknee joints in each dish; Group B, with 5 pieces (2 mm×2 mm) of the periosteum ineach dish; Group C, with 5 pieces (2 mm×2 mm×2 mm) of the cartilage in each dish; and Group D, with no addition of other tissues (control group). RNA was extracted from the cells of the cartilage explants (4 mm×4 mm×4 mm) in all the dishes. Thegene expressions of proteoglycan, collagen Ⅱ and NF-κB were defected by a reversetranscription-polymerase chain reaction (RT-PCR).Results In group A, the gene expression of proteoglycan was significantly decreased. The relative density of this gene expression had a significant difference when compared with that in group D (1.09±0.21 vs. 1.25±0.25, Plt;0.05); the gene expressions of collagen Ⅱ and NF-κB were also decreased, but they had no significant differences when compared with those in group D (Pgt;0.05). In groupB, the gene expressions of proteoglycan, collagen Ⅱ, and NF-κB were significantly increased. The relative densities of these gene expressions were 1.60±0.26, 1.57±0.24, and 4.20±2.22, respectively, which had significant differences when compared with those in group D (Plt;0.05). In group C, the relative density of the gene expression of collagen Ⅱ was 1.43±0.28, which had a significant difference when compared with that in group D (Plt;0.05), but therelative densities of the gene expressions of proteoglycan and NF-κB had no significant differences when compared with those in group D (Pgt;0.05). Conclusion The results indicate that the periosteum can up-regulate the gene expressions of proteoglycan, collagen Ⅱ and NF-κB. The NF-κB is likely to be an important nuclear transcription factor related to cartilage regeneration. The results also suggest that the periosteum maybe better in facilitating the cartilage repair and regeneration in clinical practice.
OBJECTIVE: To explore the effect of Fas/Apo-1 and Bcl-2 gene expression on mechanism of scar formation. METHODS: Immunohistochemical method was applied to defect the expression of Fas and Bcl-2 protein in fibroblasts from 10 cases with normal skin, 10 cases with hypertrophic scar and 10 cases with keloid. RESULTS: The positive expression rate of Bcl-2 protein in keloid was 83.2%, significantly higher than that in hypertrophic scar (38.6%), (P lt; 0.01), and the positive expression rate in hypertrophic scar and keloid was higher than that in normal skin (6.78%), (P lt; 0.01). But the positive expression rate of Fas/Apo-1 protein was 78.4% in normal skin 80.4% in hypertrophic scar, 84.4% in keloid respectively, which showed no significant difference among them (P gt; 0.05). CONCLUSION: Bcl-2 gene but Fas gene may take part in the formation of pathologic scar.
Objective To construct a regulatable plasmid containing single chain fusion gene of murine interleukin-12 (mIL-12) which was regulated with mifepristone (RU486) and explore its expression in vitro. Methods The p40 and p35 subunit sequence of mIL-20 were respectively obtained from the plasmid GCp35Ep40PN by polymerase chain reaction (PCR) and they were cloned into pCA14 plasmid after introducing a linker by overlap PCR. The single chain mIL-12 gene was comfirmed by sequencing and subcloned into pRS-17 vector which contains RU486 regulator cassette. The positive clone named pRS-RUmIL-12 was identified by restriction endonuclease digestion and PCR. Lipofectamine 2000 was used to transfect the pRS-RUmIL-12 to HEK293 cells followed by manufacturer’s recommendations. The protein concentration of mIL-12 induced with RU486 in supernatant of the transfected HEK293 cells was measured by ELISA. Results The sequence of single chain mIL-12 what we obtained was the same as the expected result. The results of restriction endonuclease digestion and PCR showed that the RU486-inducible regulatory vector (pRS-RUmIL-12) was successfully constructed. No significant mIL-12 protein concentration in supernatant of HEK293 cells activation was measured without the inducer RU486, whereas higher concentration of the mIL-12 protein was observed in the presence of RU486. The relationship of concentration of the mIL-12 protein and RU486 was positive correlated under definite range. Conclusion A regulatable eukaryotic expression plasmid of mIL-12 single chain fusion gene was constructed, which could be used in the further research of gene regulation and gene therapy.
Objective To observe the influences of uncoupling protein 2 (UCP-2) rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods Two UCP-2 green fluorescent protein (GFP) lentivirus constructs were created with the rs660339 locus carried C or T (UCP-2C or UCP-2T), respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control (NC) group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created. ＞80% of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased (F=29.183,P=0.001) in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two (F=15.970,P=0.004), day three (F=16.738,P=0.004), day four (F=5.414,P=0.045) post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group (F=277.138,P=0.000), and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C (P=0.003). The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group (F=425.679,P=0.000), and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C (P=0.002). The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group (F=11.827,P=0.008), while there was no difference between UCP-2T and UCP-2C group (P=0.404). Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.
Purpose
To investigate the expression of intercellular adhesion molecules ICAM-1 and Mac-1，in epiretinal membanes (ERM) of eyes wi th proliferative vitreoretinopathy (PVR)．
Methods
Twenty epiretinal membranes were obtained from eyes undergone vitrectomy for retinal detachment complicated with PVR and observed by immunohistochemical examination．
Results
Expressions of ICMA-1 and Mac-1 were observed in 18 and 15 membranes respectively．Expression of both adhesion molecules in 12 membranes．
Conclusion
The findings indicate that adhesion molecules might be involved in the development of PVR．
(Chin J Ocul Fundus Dis，2000，16：71-138)
【Abstract】ObjectiveTo detect p27 expression in rectal carcinoma and serum transforming growth factor-β1 (TGF-β1) level in these patients, and to elucidate the modulatory effect of serum TGF-β1 on p27 expression in rectal carcinoma. MethodsExpression of p27 was measured in 37 cases of rectal carcinoma, 22 of rectal adenoma and 19 of normal control specimens by immunohistochemical staining using antibodies to p27. Serum level of TGFβ1 was measured in these patients by enzymelinked immunosorbent assay (ELISA) method. Resultsp27 protein was expressed in normal rectal tissue, rectal adenoma and rectal carcinoma, and the positive rate was 89.47%, 90.91% and 64.87%, respectively. The positive rate of p27 in rectal carcinoma was significantly lower than that of normal rectal tissue and rectal adenoma (P=0.025). p27 was mainly located in nucleolus of normal rectal tissue and rectal adenoma, and the positive rate of p27 in cytoplasm of rectal carcinoma was higher than that of normal and rectal adenoma. The positives rates of serum TGF-β1 in normal group, rectal adenoma group and rectal carcinoma group were 21.05%, 27.27% and 51.35%(P=0.045),respectively. The expression of p27 related to histological differentiation, lymph node metastasis and infiltration depth. Serum level of TGF-β1 related to lymph node metastasis, infiltrated depth and CEA level. The positive rate of p27 in TGF-β1 negative group and positive group was 88.89% and 42.11%(MantelHaenszel χ2=6.755,P=0.009), respectively. ConclusionTGF-β1 may be useful in assessment of malignance and prognosis of rectal carcinoma. TGF-β1 can downregulate p27 expression in rectal carcinoma.
Objective To investigate the gene expression of beta-defensin-4 (mBD-4) and mBD-6 in acute lung injury (ALI) mouse.Methods Sixty adult mice were randomly divided into a control group and a ALI group.ALI was induced by intraperitoneal injection of lipopolysaccharide (LPS) in the ALI group.The control group was treated with same dose of normal saline.The lung tissues were harvested at different time point after stimulation.The expression of mBD-4 and mBD-6 mRNA was measured by real-time quantitative reverse transcription polymerase chain reaction.DNA sequencing was used to confirm the specificity of mBD-4 and mBD-6 cDNA fragment.Results There were no obvious mBD-4 and mBD-6 mRNA expression in mouse lung in the control group at all time points and ALI 6 h group.In the ALI group a marked increasing expression was found on 12 h，1 d and 3 d after LPS stimulation.The mBD-4 mRNA expression was significant higher in the ALI groups of 1 d and 3 d points than that of ALI 12 h group with no obvious difference between each other.There were no significant differences of mBD-6 mRNA expression between ALI groups of 12 h，1 d and 3 d points Conclusion mBD-4 and mBD-6 mRNA is not constitutive expressed in mouse lung and show a up-regulative expression pattern after ALI.
ObjectivesTo systematically review the association between the expression level of LncRNA and clinicopathological features and prognostic value of gastric cancer.MethodsWe searched PubMed, EMbase, The Cochrane Library, Web of Science, CNKI, VIP, WanFang Data and CBM databases to collect studies on the association between LncRNA overexpression and prognosis for gastric cancer from inception to April 2017. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies, then, meta-analysis was performed by using Stata 12.0 software.ResultsA total of 21 case-control studies were included. The results of meta-analysis showed that: LncRNA overexpression patients had poor TNM stage (OR=0.29, 95%CI 0.24 to 0.35, P<0.001), deeper tumor invasion (OR=0.24, 95%CI 0.12 to 0.49,P<0.001), shorter overall survival (OS) (HR=2.52, 95%CI 2.07 to 3.06,P<0.001) and disease-free survival (DFS) (HR=2.31, 95%CI 1.75 to 3.05,P<0.001).ConclusionsLncRNA overexpression is a poor prognosis risk factor for gastric cancer patients. Due to limited quantity and quality of the included studies, more high quality studies are needed to verify above conclusions.
Objective To evaluate the effect of copper-ion on the prol iferation and differentiation of human umbil ical vein endothel ial cell (HUVEC). Methods HUVEC were cultured and passaged in vitro. HUVEC were inoculated into 96-well plate with density of 5 × 103/well. All the cells were divided into 3 groups randomly according to different culture mediums: group A (5 μmol/L CuSO4), group B (25 μmol/L CuSO4), group C (control group). Every group had 4 wells, and the basic culture medium was MCDB131. The cell growth curves of 3 groups were drawn by using MTT. HUVEC were inoculated into 6-well plate with density of 2 × 105/well. Grouping of the cells was the same as the above. The gene expressions of endothel ial nitric oxide synthase (eNOS) and tyrosine kinase with immunoglobul in-l ike and EGF-l ike domain 1 (Tie-1) were detected by real-time RT-PCR. Results The growth curves revealed that the exponential growth time was the first 3 days, plateau growth time begun on the 4th day. The prol iferation of group A was ber than that of groups B and C from the 3rd day, within 2 days, the prol iferation of group B was ber than that of group C; however, it decreased and was weaker than group C from the 4th day, all showing statistically significant difference (P lt; 0.05). The results of real-time RT-PCR revealed that the expressions of eNOS in groups A, B and C were 7.294 ± 1.488, 0.149 ± 0.044 and 1.000 ± 0.253; and the expressions of Tie-1 in groups A, B and C were 1.481 ± 0.137, 1.131 ± 0.191 and 1.000 ± 0.177. Group A compared with groups B and C, both of 2 genes were up-regulated (P lt; 0.05). Group B compared with group C, eNOS was down-regulated (P lt; 0.05) and the difference of Tie-1 expression was not statistically significant (P gt; 0.05). Conclusion 5 μmol/L copper-ion can promote the prol iferation and differentiation of HUVEC effectively.
Objective
To quantify the mRNA expression of NMDAR1 gene in the retina of eyes with acute elevation of IOP in rabbit．
Methods
Tweenty-six eyes of 16 rabbits were divided into three groups: Group 1: The IOP of one eye in 10 rabbits was elevated to 60 mm Hg by ante ri or chamber infusion． Group 2: The another eye of the same rabbit in group 1 was maintained the IOP to 20 mm Hg by anterior chamber infusion． Group 3: Unilat eral eyes of six rabbits were enucleated to evaluate the mRNA levels as normal control group． PCR product was identified by Southern blotting and the mRNA expression level was quantified by RT-PCR．
Results
The results revealed no significant difference between group 1 and group 2．
Conclusion
This implies that acute elevated IOP may not affect the mRNA expression level of NMDAR1 gene．
(Chin J Ocul Fundus Dis, 2001,17:50-51)
