【Abstract】ObjectiveTo study the expressions of matrix metalloproteinase 2 (MMP2) and carbohydrate antigen 50 (CA50) in colorectal carcinoma, cancer-adjacent mucosa （2 cm from the nether edge of tumor）, cancerdistant mucosa （5 cm from the nether edge of tumor） and normal colorectal mucosa, and to elucidate their effects on the development of colorectal carcinoma. MethodsThe expressions of MMP2 and CA50 were detected immunohistochemically in 40 cases of colorectal carcinoma, cancer-adjacent mucosa, cancer-distant mucosa and 10 cases of normal colorectal mucosa. Results①The expression intensity and positive rates of MMP-2 and CA50 increased significantly in turn by normal mucosa, cancer-distant mucosa, cancer-adjacent mucosa and colorectal carcinoma. ②The expression of MMP2 was correlated with CA50 in colorectal carcinoma. ③The expression of CA50 in colorectal carcinoma was closely associated with tumor differentiation, and the expression of MMP2 in colorectal carcinoma was closely associated with differentiation and Dukes stages as well. ConclusionOver expression of MMP2 facilitates the malignant progress of colorectal carcinoma; CA50 is a reliable marker of malignance in colorectal carcinoma; CA50 and MMP2 may have synergetic effects on the development of colorectal carcinoma.
OBJECTIVE: To investigate the expression and distribution of xenoantigen in intervertebral disk of Chinese banna minipig inbred line, and to study the availability of xenograft transplantation of intervertebral disk. METHODS: Samples of intervertebral disk were collected from six Banna pigs of 8 to 11-month-old. The fixation, embedment and slice were performed. α-Gal specific binding lection (BSI-B4) were used as affinity reagents and affinity-immunohistochemistry assays (SABC methods and DAB stain) were conducted to detect the expression and distribution of xenoantigen (α-Gal). RESULTS: alpha-Gal was found in chondrocyte cell and chondrocyte-like cell in intervertebral disk which have the positive yellow-stained particulate aggradation. There was no stain in the matrix, elastic fiber and collagen fiber. CONCLUSIONS: The distribution of xenoantigen is locally in the tissue of intervertebral disk and its expression is weak. This suggests that the intervertebral disk of Banna pig may be alternative donor for xenotransplantation.
Radioimmunoassay was performed to measure carcinoembryonic antigen (CEA) levels in gastric juice before and after operation in 51 gastric cancer patients (group Ⅰ)， 33 patients with gastric benign lesion (group Ⅱ) and 8 patients with malignant lesion in digestive system other than gastric cancer (group Ⅲ). The results showed that preoperative CEA levels of in group Ⅰ were the highest among three groups (Ｐ＜0.01), but no statistic difference was noted in group Ⅱ and group Ⅲ. In group Ⅰ and group Ⅱ, postoperative CEA levels were higer than the preoperative levels. The authors believe that preoperative CEA measurement of gstric juice is an accessory method in diagnosing gastric cancer, nevertheless, there is no diagnostic significence of postoperative measurement in patient undergone partial gastrectomy.
Objective To observe the inhibitory effects of local co-transfection of tissuetype plasminogen activator(tPA) gene and proliferating cell nuclear antigen antisense oligodeoxynucleotides(PCNA-ASODN) on the intima proliferation and restenosis of autograft artery in rabbits. Methods One hundred and twenty male Zelanian rabbits were randomly divided into four groups(n=30, in each group): control group, PCNA-ASODN group, tPA group and tPA+PCNAASODN group. The left and right external iliac arteries (length 1.0 cm) were transplanted reciprocally. The transplanted arteries were respectively soaked in lipofection, PCNAASODN, pBudCE4.1/tPA and pBudCE4.1/tPA+PCNA-ASODN solution about 15 minutes. The transplanted arteries were sutured with 9-0 sutures soaked in PCNA-ASODN and pBudCE4.1/tPA solution. Each group were divided into five subgroups(n=6, in each subgroup) according to the sacrifice time (3 d, 7 d, 14 d, 28 d and 56 d after operation). On every sacrifice time point, the vascular specimens were harvested. The thrombocyte assembling and thrombus forming lining vessel wall were observed by scanning electron microscope. The pathological morphology of transplanted arteries were observed under microscope(HE). The intimal areas and stenosis ratio(%) of transplanted arteries were calculate and analyzed statistically among groups by computer system. The mRNA expression of tPA gene in transplanted ressel wall was detected with vevere transcriptionPCR(RT-PCR). The number of PCNA positive cells in transplanted vessel wall was counted by SP immunochemisty.Results The mRNA expression of tPA gene in the transplanted vessel wall in tPA and tPA+PCNA-ASODN groups was higher than that of the other two groups(P＜0.01).The number of PCNA positive cells in the transplanted arteries in PCNAASODN, tPA and tPA+PCNAASODN groups were significantly lower than that of control group(P＜0.05，P＜0.01). The intimal areas and degrees of luminal stenosis of PCNAASODN, tPA and tPA+PCNAASODN groups were lower than those of control group(P＜0.05，P＜0.01), and those of tPA＋ PCNA-ASODN group were lower than those of PCNA-ASODN and tPA groups(P＜0.05). Scanning electron microscopy showed that there were a few thrombocytes lining the vessel wall of tPA group and tPA+PCNAASODN group and no thrombus, whereas there were abundant thrombocytes and thrombi lining the vessel wall of the control group. Conclusion Co-transfection of tPA gene and PCNA-ASODN can effectively inhibit the proliferation of VSMC, hyperplasia of intima and restenosis of transplanted artery.
Objective To investigate the effects of recombinant adenovirus-mediated co-transfection of carcinoembryonic antigen (CEA) gene and erythropoietin (EPO) gene on promoting hematopoietic stem cells directly producing erythrocyte vaccine against colon cancer. Methods The expression adenovirus vectors carrying CEA and EPO or green fluorescent protein (GFP) gene were constructed respectively, and recombinant adenovirus carrying CEA, EPO or GFP were packaged and produced respectively. The bone marrow-derived mesenchymal stem cells (MSCs) of mice were isolated and cultured in vitro by anti-CD117 magnetic bead separation, and were transfected with CEA (CEA group), EPO (EPO group) or GFP (blank vector group), co-transfected with CEA and EPO (CEA-EPO group). The expressionsof CEA and EPO gene and its protein after transfection in supernatant fluid of culture were detected by realtime-PCR and Western blot method in each group. We had checked and obtained the vaccine with co-transfection of CEA gene and EPO gene by cell red line marker antibody CD71 and GPA, then we carried on experiments with the vaccine in vitro and in vivo. There were 4 groups in our trail: blank vector group, CEA group, EPO group, and CEA-EPO group. Results We had successfully gathered the hematopoietic stem cells, flow cytometry analysis result showed that there were significant differences before and after purification for positive selected samples (P<0.05). The expressions of double genes (CEA-EPO gene) and protein showed CEA-EPO gene were successfully transfected into the hematopoietic stem cells. We had confirmed erythrocyte vaccine with co-transfection of CEA and EPO gene by antibody CD71 and GPA with flow cytometry. The monocytes cytotoxicity on colon cancer cell line CT26 showed that lysis of target cells of CEA-EPO group were higher than those of other 3 groups when in proportion of 40∶1 (P<0.05). In the experimentation of neoplasma format, the volume of tumor and mortality were smaller or lower, but survival time was longer of CEA-EPO group in2 weeks after treatment (P<0.05). Conclusions The erythrocyte vaccine with co-transfection of CEA gene and EPO gene has efficient anti-tumor effects on colon cancer. Not only can promote hematopoietic stem cell directly producing erythrocyte vaccine, but also can produce tumor antigen vaccine against colon cancer.
The expression of T antigen in rectal cancer and mucosa remote from carcinoma by immunohistochemistry was investigated. Mucin protein was also examined by HID-AB staining. The results showed that the expression of T antigen in rectal cancer was much ber than those in 10cm mucosa remote from carcinoma and no significant difference as compared with 5cm mucosa. The sialomucin reactions in 5cm and 10cm mucosa remote from carcinoma were 45% and 20% respectively. The coincident sialomucin positive reaction and expression of T antigen were found in 40% 5cm remote mucosa .There is significant correlation between them (Ｐ＜0.05). The authors conclude that the expression of tumorrelated antigen and change of mucin protein in remote mucosa without malignant invasion may suggest the malignant potential of the mucosa. Further investigations should be performed into the effect of these changes on the local recurrence after redical resection of rectal cancer.
ObjectiveTo investigate the expression changes and the repair effect of mitogen and stress- activated protein kinase 1 (MSK1) on spinal cord injury (SCI) in rats.MethodsOne hundred and twenty male Sprague Dawley (SD) rats (weighing 220-250 g) were used for the study, 70 of them were randomly divided into sham-operation group and SCI group (n=35), the rats in SCI group were given SCI according to Allen’s method, and the sham-operation group only opened the lamina without injuring the spinal cord; spinal cord tissue was collected at 8 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, and 7 days after invasive treatment, each group of 5 rats was used to detect the expression of MSK1 and proliferating cell nuclear antigen (PCNA) by Western blot assay. Another 20 SD rats were grouped by the same method as above (n=10). In these rats, a negative control lentiviral LV3NC dilution was injected at a depth of approximately 0.8 mm at the spinal cord T10 level. The results of transfection at 1, 3, 5, 7, and 14 days after injection were observed under an inverted fluorescence microscope to determine the optimal transfection time of the virus. The other 30 SD rats were randomly divided into group A with only SCI, group B with a negative control lentiviral LV3NC injected after SCI, and group C with MSK1 small interfering RNA (siRNA) lentivirus injected after SCI, with 10 rats each group. The Basso, Beatlie, Bresnahan (BBB) score of hind limbs was measured at 1, 3, 5, 7, and 14 days after treatment; spinal cord tissue collected at the optimal time point for lentivirus transfection was detected the expression changes of MSK1 and PCNA by Western blot and the localization by immunofluorescence staining of MSK1 and PCNA proteins.ResultsWestern blot assay showed that there was no significant changes in the expression of MSK1 and PCNA at each time points in the sham-operation group. In the SCI group, the expression of MSK1 protein was gradually decreased from 8 hours after injury to the lowest level at 3 days after injury, and then gradually increased; the expression change of PCNA protein was opposite to MSK1. The expression of MSK1 in SCI group was significantly lower than that in the sham-operation group at 1, 2, 3, and 5 days after injury (P<0.05), and the expression of PCNA protein of SCI group was significantly higher than that of the sham-operation group at 8 hours and 1, 2, 3, 5, and 7 days after injury (P<0.05). The fluorescence expression of both the SCI group and the sham-operation group has be found and peaked at 7 days. There was a positive correlation between fluorescence intensity and time in 7 days after transfection. With the prolongation of postoperative time, the BBB scores of groups A, B, and C showed a gradually increasing trend. The BBB score of group C was significantly lower than those of groups A and B at 5, 7, and 14 days after treatment (P<0.05). After transfection for 7 days, Western blot results showed that the relative expression of MSK1 protein in group C was significantly lower than that in groups A and B (P<0.05); and the relative expression of PCNA protein was significantly higher than that in groups A and B (P<0.05). Immunofluorescence staining showed that MSK1 was expressed in the nuclei of the spinal cord and colocalized with green fluorescent protein, neuronal nuclei, and glial fibrillary acidic protein (GFAP). The relative expression area of MSK1 positive cells in group C was significantly higher than that in group B (P<0.05), and the relative expression areas of PCNA and GFAP positive cells were significantly lower than those in group B (P<0.05).ConclusionLentivirus-mediated MSK1 siRNA can effectively silence the expression of MSK1 in rat spinal cord tissue. MSK1 may play a critical role in the repair of SCI in rats by regulating the proliferation of glial cells.
Objective Epidermal stem cells (ESCs) can actively partici pate in wound heal ing and enhance reepithel ial ization. To establ ish ideal diabetes mell itus (DM) rat models and to investigate the expression of keratin 19 (K19),β1-integrin, β-catenin, and prol iferating cell nuclear antigen (PCNA) in ESCs of DM rat model, then to study the potential mechanism of difficult recovering wounds of diabetic skin. Methods Twenty male SD rats (weighing 250-300 g) were dividedinto DM group and normal control group randomly (n=10). The DM rat model was made by intraperitoneal injected 65 mg/kg streptozocin (STZ), the normal control group was not treated. At 4 weeks after injection, pancreatic tissue was harvested for HE staining in two groups. The ESCs isolated from full-thickness skins of the back of two group rats were culutured and identified. The 2nd passage of ESCs were obtained for immunocytochemical staining of K19, β1-integrin, β-catenin, and PCNA. Meanwhile, the cell cycle were measured by flow cytometry. The cell colony formation rates were detected after 1 week. Results The achievement ratio of DM rat model was 90% with good stabil ity. HE staining showed that the number of islet cells significantly decreased with degeneration and necrosis in DM group; the structure of islet cell was clear without degeneration and necrosis in normal control group. The integral absorbance values of positive expression for K19, β1-integrin, β-catenin, and PCNA in ESCs of DM group (82.63 ± 14.77, 21.59 ± 4.71, 6.49 ± 6.58, and 90.77 ± 12.44, respectively) were significantly lower than those in the normal control group (151.24 ± 42.83, 54.48 ± 17.43, 116.39 ± 9.26, and 110.62 ± 20.67, respectively) (P lt; 0.01). The clone forming efficiency of ESCs in DM group (6.43% ± 1.01% ) was significantly lower than that in the normal control group (11.37% ± 1.62%) (P lt; 0.01). Flow cytometry indicated that 88.89% of cultured ESCs in the DM group were in resting state/ pre-DNA-synthetic gap (G0/G1), and the apoptosis rate was 3.98%; 91.50% in the normal control group and the apoptosis rate was 0. Conclusion The DM rat model can be effectively induced by intraperitoneal injected 65 mg/ kg STZ. The decreased amount and the low prol iferation and differentiation capacity of ESCs may be one of the important mechanisms of difficult recovering wounds of DM rats.
Objective To investigate the relationship between human leukocyte antigen(HLA)-B51 and Behcet′s disease (BD) with uveities. Methods HLA-A and HLA-B antigen of 27 pateints with BD and 30 healthy persons were detected by microly mphocyte toxicity asssay. HLA-B51 allele (HLA-B5101-B5107) in BD patients with positive HLA-B51 antigen and the controls was detected by polymerase chain reaction-sequence specific primer(PCR-SSP). Results The positive rate of HLA-B51 was 63% and 16.7% in BD patients and the controls, respectively (χ2=12.9, P＜0.001, Pc＜0.05,RR=8.5). Uveities was found in 11 out of 27 BD patients with uveitis. No relativity was found between HLA-B51 and uveitis in BD patients(RR=2.07,χ2=0.759,P＞0.25),and weak association was found between HLA=B5101 and uveitis (RR=2.67, χ2=1.395, P＞0.10). Conclusions HLA-B51 might be a susceptible gene for BD, and there was a weak association between HLA-B51(HLA-B5101) and BD patients with uveitis.(Chin J Ocul Fundus Dis，2004，20：203-205)
ObjectiveTo explore value of preoperative neutrophil-to-lymphocyte ratio (NLR) combined with carbohydrate antigen 19-9 (CA19-9) in evaluating prognosis for patients with pancreatic head carcinoma.MethodsThe clinical data of patients with pancreatic head carcinoma underwent radical pancreaticoduodenectomy in the Lanzhou University Second Hospital from February 2011 to August 2017 were retrospectively analyzed. The preoperative serum albumin (ALB), CA19-9, NLR, carcino-embryonic antigen (CEA), alpha-fetoprotein (AFP), N stage, tumor differentiated degree, and other indicators were recorded and the patient’s overall survival was recorded. Linear correlation analysis was used to examine the association of NLR with serum CA19-9. The receiver operating characteristic (ROC) curve was used to calculate the cutoff values of NLR and CA19-9 corresponding to the overall survival rate of 2-year. Kaplan-Meier method was used for survival analysis. Cox proportional hazards regression model was used to evaluate the prognostic value of preoperative CA19-9 and NLR.Results① A total of 121 patients were enrolled in this study. There was no significant correlation between the preoperative serum CA19-9 and NLR (r=0.100 05, P=0.272 9). ② Multivariate analysis showed that higher NLR, higher CA19-9 level, higher N stage, and lower tumor differentiation were the risk factors for overall survival of pancreatic head carcinoma (P<0.05). ③ The cutoff values of CA19-9 and NLR in evaluating the 2-year overall survival rate were 123.3 U/mL and 2.34 respectively by the ROC curve analysis, the area under ROC curve of CA19-9, NLR, and CA19-9 combined with NLR were 0.763, 0.686, and 0.823, respectively. ④ The proportions of patients with CEA≥5 μg/L and ≥N1 stage were higher in the patients with CA19-9≥123.3 U/mL as compared with patients with CA19-9<123.3 U/mL (P<0.05); The proportions of patients with preoperative serum ALB level <35 g/L and CEA≥5 μg/L, ≥N1 stage, and poor differentiation were higher in the patients with NLR≥2.34 as compared with the patients with NLR<2.34 (P<0.05). ⑤ Kaplan-Meier survival analysis showed that patients with CA19-9≥123.3 U/mL and NLR≥2.34 had worse overall survival than those patients with CA19-9<123.3 U/mL and NLR<2.34 (HR=3.599, P<0.01; HR=2.506, P<0.01). The patients with CA19-9≥123.3 U/mL and NLR≥2.34 (n=42), CA19-9≥123.3 U/mL or NLR≥2.34 (n=48), CA19-9<123.3 U/mL and NLR<2.34 (n=31) had better overall survival in turn (overall and between groups, all P<0.01).ConclusionPreoperative serum CA19-9 combined with NLR has an important value in evaluating prognosis for patients with pancreatic head carcinoma.