Objective To resolve the tough problem of how to observe the growing cells in an opaque vector. Methods The urethral epithelial cells from a young male New Zealand rabbit were inoculated, and were primarily cultured in vitro and subcultured for 3 passages. Then, the urethralepithelial cells were cultured in the collagen chitosan complex for 3, 7, 14 and 21 days. The cells were dyed with 6-carboxyfluorescein diacetateacetoxymethyl ester and propidium iodine, respectively. Then, Interactive Laser Cytometer was used to detect the growing cells. Results The urethral epithelial cells grew and proliferated very well in the collagen chitosan complex vector. After the urethral epithelial cells grew in the collagen-chitosan complex vector for 3 and 7 days, the fluorescent density amount of the surviving cells were(1.09±0.13)×10.8 and (2.04±0.13)×10.8, respectively. However, after 14and 21 days, the fluorescent density amount of the surviving cells was (0.55± 0.09)×10.8 and (0.47±0.03)×108, respectively. There was a significant difference when compared with the amount of the surviving cells at 3 and 7 days（P＜0.05）.Conclusion Using Interactive Laser Cytometer for measurement of the green and red fluorescent densities of different waves, the activity of the cultured urethral epithelial cells in vitro can be rapidlymeasured with the in situ quantitation method. This method solves a difficult problem of observing the growing cells in an opaque vector. The dynamic growing state of the engineering tissues can be observed.
ObjectiveTo investigate the expression and relation of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in rats with diabetic retinopathy.MethodFifty-five Wistar rats were randomly divided into the control group (10 rats), and 1, 3, and 5-month-diabetes group (15 rats in each diabetes group), and the diabetic models were set up. The expressions of VEGF and bFGF were detected by situ hybridation and immunohistochemistry on retinal paraffin sections.ResultsThe results of situ hybridation showed that expression of bFGF was found in 3-month-deatbtes group with the percentage of 77.8%, and 88.9% in 5-month-deatbtes group; the positive expression of VEGF was not found in 3-month-deatbtes group but in 5-month-deatbtes group with the percentage of 66.7%. Immunohistochemistry indicated that the positive expression of bFGF started in 3-month-deatbtes group with the percentage of 55.6%, and 88.9% in 5-month-deatbtes group; the percentage of the expression of VEGF was 33.3% in 3-month-deatbtes group and 88.9% in 5-month-deatbtes group.ConclusionThe expression of VEGF occurs after the expression of bFGF in rats with DR.(Chin J Ocul Fundus Dis, 2005,21:37-40)
Objective To compare the outcome of uterine exteriorisation repair with in situ in caesarean section. Methods A randomized controlled trial with 220 cases were recruited. Woman with term singleton pregnancy underwent caesarean section and without severe complication were randomly allocated to the two groups (112 cases in exteriorisation group and 108 cases in situ group). Women in treatment group received exteriorisation when the uterus was sutured, While others had the uterus repaired in situ as control. Main outcome measures included perioperative haemodynamic parameters, loss of blood, changes in haemoglobin concentration, duration of operation, postoperative pain score and febrile days, gastrointestinal discomforts and function recovery as well as postoperative maternal morbidity.Data were analyzed by SPSS 11.0. Results Haemoglobin concentration dropped in both groups after caesarean section, and the drops in control group was significantly higher than that of treatment group (t=-2.902, P=0.004). In both groups, pulse before operation was markedly higher than when suturing the uterus and postoperation (Plt;0.05), but no difference was observed between the two groups. Systolic blood pressure of treatment group was lower than that of control group before operation, when suturing the uterus and after operation (F=5.246, P=0.022), but there was no difference among these 3 time points within the group. Onset of flatus was earlier in control group than in treatment group (t=5.567, P=0.000). No difference was identified between the two groups when receiving the different suturing methods. No severe maternal morbidity was observed.Conclusions Uterine exteriorisation and in situ repair have similar effects on intra- and postoperative outcomes. In routine process.
Objective To explore the expression of tumor necrosis factor (TNF) mRNA, TNF and TNFR in the gallbladder mucosa which developed from hyperplasia, dysplasia to carcinoma, and to further discuss the relationship between TNF and pathogenesis of gallbladder carcinoma. Methods In situ hybridization and immunohistochemistry were used to determine TNF mRNA, TNF protein and TNFR protein expression in hyperplasia, dysplasia and carcinoma of gallbladder. Results ①No one of 20 cases of gallbladder hyperplasia was found to express TNF mRNA, while 4 of 20 (20%) cases of dysplasia and 18 of 20 (90%) cases of carcinoma were found to express TNF mRNA (P＜0.05). ②For the expression of TNF mRNA in mononuclear cells (MNC), positive staining was found in 15% of gallbladder hyperplasia, 85% of dysplasia and 90% of carcinoma, respectively (P＜0.05). The cell numbers of positive staining MNC were 4.85±1.50, 6.00±2.71 and 9.33±3.07, respectively (P＜0.05). ③In gallbladder carcinoma, the cell number of carcinoma and MNC with positive TNF mRNA expression was correlated with clinical stage (P＜0.05). The higher the clinical stage, the more the positive staining cell numbers. The positive staining cell numbers of carcinoma in stage Ⅰ-Ⅲ and Ⅳ-Ⅴ were 9.13±4.39 and 14.80±4.02, respectively (P＜0.01), and the positive staining cell numbers of MNC were 7.13±2.53 and 11.10±2.23, respectively (P＜0.05). ④The cell numbers of carcinoma and MNC with TNF mRNA expression increased with tumor size. In tumors with diameter over 2 cm and less than 2 cm, the positive staining cell numbers of carcinoma were 14.00±4.20 and 8.83±4.96, respectively (P＜0.05), and that of MNC were 10.50±2.54 and 7.00±2.83, respectively (P＜0.05). ⑤The region of TNF protein expression was similar to that of TNF mRNA, but TNF protein expression was more frequent and wider than that of TNF mRNA. ⑥The tumor necrosis factor receptor was expressed in tumoral vascular endothelial cells and MNC in all cases of carcinoma, but was negatively stained in mucosa epithelial cells and tumor cells of all cases. ⑦There was positive linear correlation in TNF mRNA between tumor cell and MNC (r=0.687, P＜0.01), same as that in TNF protein expression (r=0.742, P＜0.01); and there was positive linear correlation in tumor cell between TNF mRNA and TNF protein expression (r=0.847, P＜0.01), same as that in MNC (r=0.643, P＜0.01). Conclusion The TNF mRNA and TNF protein expression are increasing during the development of gallbladder mucosa epithelial from hyperplasia, dysplasia to carcinoma, and increasing with tumor stage. It suggests that TNF may contribute to carcinogenesis of gallbladder carcinoma induced by gallstone, and related to the progression of gallbladder carcinoma.
Objective To study the expression and its clinical significance of p16 in human gastric carcinomas. MethodsThe expression of p16 protein and mRNA in human gastric carcinomas using SP immunohistochemical and in situ hybridization (ISH) methods were examined. Results Of the 85 cases tested, 65.88% (56 cases) showed positive staining of p16 protein in the primary lesions. The positive rate of p16 protein was significantly lower in the cases with deep invasion, poor differentiation or shorter survival periods (Ｐ＜0.05). The positive rate of p16 mRNA expression in human gastric carcinomas was 47.37% (in 38 cases). Conclusion p16 gene may correlate with the development and progress of gastric carcinomas. The expression of p16 gene may be a useful tool for showing biological behavior and prognosis of human gastric carcinomas.
Objective To investigate the association of the expression of CD15 mRNA with the invasion and prognosis of hepatocellular carcinoma (HCC) and the expression of nm23H1 mRNA. Methods In situ hybridization and immunohistochemistry methods were used to detect the expression of CD15 mRNA and protein nm23H1 mRNA in HCC.Results In 99 cases of HCC, the positive rate of CD15 mRNA,its protein and nm23H1 mRNA were 38.4%, 36.4% and 76.8%, respectively. The expression of CD15 mRNA was consistent with its protein and negatively correlated with the expression of nm23H1 mRNA. The expression of CD15 mRNA and its protein, nm23H1 mRNA were associated with the invasiveness and metastasis of HCC and the prognosis of HCC patients. Conclusion The detection of CD15 expression could be a new pathological biology index to judge the metastasis and prognosis of HCC.
Objective To investigate the influnce of L-arginine (L-Arg) and L-nitro-arginine-methyl-ester(L-NAME) to purified retinal ganglion cells(RGCs) apoptosis of rats cultured in different consistencies of L-Arg and L-NAME. Method RGCs from Sprague Dawley (SD) neonatal rats(postnatal 1~5 day) were cultured in assimilative culture solution in vitro and RGCs were purified by Thy1.1 with sheep anti rat FITC monoclonal antibody. RGCs were cultured in different consistencies of L-Arg and L-NAME: 1×10-6, 1×10-5,1×10-4, 1×10-3, 1×10-2 and 1×10-1 mol/L for 24 hours and 48 hours, respectively. The changes of bcl-2, bax and p53 mRNA in RGCs in different consistencies of L-Arg and L-NAME were demonstrated qualitatively and quantitatively by in situ hybridization, and their apoptosis were detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) method, respectively. Results After 24 hours in vitro, the purification rate of RGCs in the experiment arrived at 97 %. After 48 hours, there were a few apoptotic cells expression in the control group. Apoptotic cells expression in L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L groups increased that had a significant difference with the control group (Plt;0.05). In the group of L-Arg≥1×10-3 mol/L and L-NAME≥1×10-1 mol/L, the expression of bcl-2 mRNA in RGCs became weaker and weaker as the consistencies were increased, but the expression of bax and p53 mRNA in RGCs became higher and higher and had a significant difference with control group (Plt;0.05). Conclusion Lower concentration of L-Arg can promote the growth of purified RGCs in vitro and higher concentration of L-Arg can promote the apoptosis of RGCs. (Chin J Ocul Fundus Dis, 2002, 18: 137-139)
Objective To fabricate in situ crosslinking hyaluronic acid hydrogel and evaluate its biocompatibility in vitro. Methods The acrylic acid chloride and polyethylene glycol were added to prepare crosslinking agent polyethylene glycol acrylate (PEGDA), and the molecular structure of PEGDA was analyzed by Flourier transformation infrared spectroscopy and 1H nuclear magnetic resonance spectroscopy. Hyaluronic acid hydrogel was chemically modified to prepare hyaluronic acid thiolation (HA-SH). And the degree of HA-SH was analyzed qualitatively and quantitatively by Ellman method. HA-SH solution in concentrations (W/V) of 0.5%, 1.0%, and 1.5% and PEGDA solution in concentrations (W/V) of 2%, 4%, and 6% were prepared with PBS. The two solutions were mixed in different ratios, and in situ crosslinking hyaluronic acid hydrogel was obtained; the crosslinking time was recorded. The cellular toxicity of in situ crosslinking hyaluronic acid hydrogel (1.5% HA-SH and 4% PEGDA mixed) was tested by L929 cells. Meanwhile, the biocompatibility of hydrogel was tested by co-cultured with human bone mesenchymal stem cells (hBMSCs). Results Flourier transformation infrared spectroscopy showed that most hydroxyl groups were replaced by acrylate groups; 1H nuclear magnetic resonance spectroscopy showed 3 characteristic peaks of hydrogen representing acrylate and olefinic bond at 5-7 ppm. The thiolation yield of HA-SH was 65.4%. In situ crosslinking time of hyaluronic acid hydrogel was 2 to 70 minutes in the PEGDA concentrations of 2%-6% and HA-SH concentrations of 0.5%-1.5%. The hyaluronic acid hydrogel appeared to be transparent. The toxicity grade of leaching solution of hydrogel was grade 1. hBMSCs grew well and distributed evenly in hydrogel with a very high viability. Conclusion In situ crosslinking hyaluronic acid hydrogel has low cytotoxicity, good biocompatibility, and controllable crosslinking time, so it could be used as a potential tissue engineered scaffold or repairing material for tissue regeneration.
Objective To observe the expression of proinflammatory factors messenger RNA(mRNA) in periretinal membrane of proliferative vitreoretinopathy. Methods Fourteen specimens of periretinal membrane were collected during vitrectomy, and they were made into paraffin sections.The presence of mRNA coding for IL-1,IL-6,IL-8 and TNF alpha was observed by in situ hybridization(ISH) with biotin labeled oligonuclotide probes respectively.The eyeball after corneal grafting was made as normal control. Results No expression of proinflammatory factors mRNA was found in normal human retina.Positive staining was present in 5 specimens.In these specimens, IL-1βmRNA was found in 3 specimens and TNFαmRNA was found in 3 specimens,there is 1 specimen expressed IL-8 mRNA and 3 specimens expressed IL-6 mRNA.In these positive specimens, one contained cells expressing mRNA for IL-1βbeta and IL-6, and one exhibited cells expressing mRNA for IL-1β、IL-8 and TNFα,two membranes possessed positive cells for IL-6 and TNFαmRNA, one membrane contained IL-1βmRNA positive cells only. Conclusion These findings suggest that these cytokines may be locally produced by cells infiltrating epiretinal membranes. The expression of IL-1β, IL-6, IL-8 and TNFαmRNA within retinal membranes provides further evidence of a pathogenic role of these cytokines in proliferative vitreoretinopathy. (Chin J Ocul Fundus Dis, 2002, 18: 286-288)
ObjectiveTo review the current research status of in situ three-dimensional (3-D) printing technique and future trends. MethodsRecent related literature about in situ 3-D printing technique was summarized, reviewed, and analyzed. ResultsBased on the cl inical need for surgical repair, in situ 3-D printing technique is in the preliminary study, mainly focuses on in situ dermal repair and bone and cartilage repair, and succeeds in experiments, but there are still a lot of problems for cl inical application. ConclusionWith the development of in situ 3-D printing technique, it will provide patients with real-time and in situ digital design and 3-D printing treatment with a timely and minimally invasive surgical repair process. It will be widely used in the future.