Objective To study the vascularization of the compositeof bone morphogenetic protein 2 (BMP-2) gene transfected marrow mesenchymal stem cells (MSCs) and biodegradable scaffolds in repairing bone defect. Methods Adenovirus vector carrying BMP-2 (Ad-BMP-2) gene transfected MSCs and gene modified tissue engineered bone was constructed. The 1.5 cm radial defect models were made on 60 rabbits, which were evenly divided into 4 groups randomly(n=15, 30 sides). Different materials were used in 4 groups: Ad-BMP-2 transfected MSCs plus PLA/PCL (group A), AdLacz transfected MSCs plus PLA/PCL (group B), MSCs plus PLA/PCL (group C) and only PLA/PCL scaffolds (group D). The X-ray, capillary vessel ink infusion, histology, TEM, VEGF expression and microvacular density counting(MVD) were made 4, 8, and 12 weeks after operation. Results In group A after 4 weeks, foliated formed bones image was observed in the transplanted bones, new vessels grew into the bones, the pores of scaffolds were filled with cartilage callus, osteoblasts with active function grew around the microvessels, and VEGF expression and the number of microvessels were significantly superior to those of other groups, showing statistically significant difference (Plt;0.01); after 8 weeks, increasingly more new bones grew in the transplanted bones, microvessels distended and connected with each other, cartilage callus changed into trabecular bones; after 12 weeks, lamellar bone became successive, marrow cavity recanalized, microvessels showed orderly longitudinal arrangement. In groups B and C, the capability of bone formation was weak, the regeneration of blood vessels was slow, after 12 weeks, defects were mostly repaired, microvessels grew among the new trabecular bones. In group D, few new vessels were observed at each time, after 12 weeks, broken ends became hardened, the defectedarea was filled with fibrous tissue. Conclusion BMP-2 gene therapy, by -upregulating VEGF expression, indirectly induces vascularization ofgrafts,promotes the living of seed cells, and thus accelerates new bone formation.
【 Abstract 】 Objective To investigate the effects of 250 ml/m3 carbon monoxide (CO) inhalation or intraperitoneal infusion on lipopolysaccharide (LPS) induced rat intestinal tract injury, and to detect the roles of p38 mitogen-activated protein kinase (MAPK) pathway during CO administration. Methods After received 5 mg/kg LPS or an equal volume of normal saline by intravenous injection, 108 male SD rats were randomly divided into 6 groups: control group, CO inhalation (250 ml/m3) group, CO intraperitoneal infusion (250 ml/m3 at a rate of 2 L/min) group, LPS (5 mg/kg) group, LPS (5 mg/kg)+CO inhalation (250 ml/m3) group and LPS (5 mg/kg)+CO intraperitoneal infusion (250 ml/m3 at a rate of 2 L/min) group. The animals were differently sacrificed at 1, 3 and 6 h for the observation, and the ileum tissues were homogenized for determination the levels of platelet activator factor (PAF), intercellular adhesion molecule-1 (ICAM-1) and interlukin-10 (IL-10) with enzyme-lined immunosorbent assay, the content of maleic dialdehyde (MDA) with thiobarbitric acid, the activity of myeloperoxidase (MPO) with chemical method, the activity of superoxide dismutase (SOD) with hydroxylamine, the activity of phosphorylated p38 MAPK with Western blot, the pathology with light microscope, and the extents of cell apoptosis were showed by the ratio of the apoptotic cells which had less DNA to the total cells of a cell-suspension sample by using the flow cytometry after being stained with propidium iodide. Results Compared with both control, CO inhalation and intraperitoneal infusion group at the same time point, the levels of PAF, ICAM-1, MDA, MPO, cell apoptosis rate and the phosphorylated p38 MAPK protein in LPS group were increased, while IL-10 and SOD were decreased (P ＜ 0.05 or 0.01), and accompanied by severe intestinal tract injury. There were no statistics differences at the different time point in the same group. PAF, ICAM-1, MDA, MPO and cell apoptosis rate in both LPS+CO inhalation group and LPS+CO intraperitoneal infusion group were lower, while IL-10 and SOD were higher than the corresponding value in LPS group at the same time point (all P ＜ 0.05), with ameliorate injury too, but the expression of phosphorylated p38 MAPK was further up-regulated than that of LPS group (all P ＜ 0.05). However, there were no significant differences in these parameters between LPS+CO inhalation group and LPS+CO intraperitoneal infusion group. Conclusion 250 ml/m3 CO inhalation and intraperitoneal infusion exerts the similar protection against LPS induced rat intestinal tract injury via anti-oxidant, anti-inflammation, and anti-apoptosis. This may involve the p38 MAPK pathway.
Objective To evaluate the host immune reaction against adenovirus mediated human bone morphogenetic protein 2 (Adv-hBMP-2) gene therapy in repairof tibial defects. Methods Twelve goats were made 2.1 cm segmental defects in he tibial diaphysis and divided into 2 groups. AdvhBMP2 transfected marrow mesenchymal stem cells(MSCs) and untransfected MSCs were implanted into the defect sites of transfected group(n=7) and untransfected group (n=5), respectively. The defect repair was observed by X-ray films after 4, 8, 16 and 24 weeks of transplantation and cellular and humoral immune reactions to adenovirus were assayed before implantation and after implantation. Results More bony callus was found in the bone defects of transfected group. The healing rates were 6/7 in transfected group and 2/5 in untransfected group, respectively at 24 weeks after implantation. The mixed culture of lymphocytes and MSCs showed that the lymphocytes stimulation indexes (SI) increased 14 days after implantation, and there was significant difference between the transfected group (4.213±1.278) and the untransfected group(－0.310±0.147，Plt;0.05); SI decreased after 28 days, but there was no significant difference between the transfected group (2.544±0.957) and the untransfected group (3.104±0.644，Pgt;0.05). After 14, 28, 49, and 120 days of treatment, the titer values of neutralizing antibody against Adv-hBMP-2 (log0.1) were 2.359±0226, 2.297±0.200, 2.214±0.215 and 2.297±0.210 in transfected group, and -0.175±0.335, -0.419±0.171, 0±0.171 and 0.874±0.524 in untransfected group, being significant differences betweentwo groups(Plt;0.05). Conclusion Adenovirus mediated BMP-2gene therapy can cause cellular and humoral immune reactions against adenovirus, which can eliminate the influence of adenoviral genes and proteins within a certain period.
Objective To explore the effect of age and gene therapyon the differentiation of marrow mesenchymal stem cells （MSCs） of the rats. Methods MSCs from the young (1-month-old), adult (9-month-old), and the aged(24monthold) rats were expanded in culture and infected with adenovirus mediated human bone morphogenetic protein 2 gene (Ad-BMP-2). The expression of BMP-2 and osteoblastic markers such as alkaline phosphatase(ALP), collagen Ⅰ(Col Ⅰ), bone sialoprotein(BSP) and osteopontin(OPN) were assayed during the process of differentiation. Their abilities to induce ectopic bone formation in nude mice were also tested. Results There was no significant difference in the expression of BMP-2 among the 3 groups. ALP activity assay and semi-quantitative reverse transcription polymerase chain reaction(RT-PCR) demonstrated that there were no significant differences in the expression of osteoblastic markers ALP, Col-Ⅰ, OPN and BSP amongthe 3 groups. Histomorphometric analysis indicated that there were no significant differences in the volume of the newly formed ectopic bones in nude mice amongthe 3 groups. Conclusion MSCs obtained from the aged ratscan restore their osteogenic activity following human BMP-2 gene transduction, therefore provides an alternative to treating the aged bone disease.
摘要:目的: 探讨益活清下法治疗重症急性胰腺炎(severe acute pancreatitis, SAP)对血清单核趋化蛋白1及对器官功能不全的影响。 方法 : 依据纳入和排除标准,选取SAP患者24例,按1︰1随机分为治疗组和对照组,在接受相同西医治疗的基础上,治疗组使用中药“益活清下”法治疗,对照组同时接受中药安慰剂治疗。测定患者第0、1、3、5、7天血清MCP1的浓度水平,比较各器官功能不全的发生率与持续时间。 结果 :两组入院时Rason评分、CT评分、急性生理和慢性健康评价指标Ⅱ评分无统计学差异(〖WTBX〗P gt;005)。对照组第3天MCP1浓度水平明显高于治疗组,差异有统计学意义(〖WTBX〗P lt;005),对照组肠、肝功能不全的发生率高于治疗组,持续时间长于治疗组,但无统计学差异(〖WTBX〗P gt;005)。 结论 :益活清下法治疗重症急性胰腺炎,可降低患者血清MCP1的水平。Abstract: Objective: To investigated the impact of Yihuo Qingxia method on the serum monocyte chemoattractant protein1 of severe acute pancreatitis (SAP)and on the organs disfunction. Methods : Twentyfour SAP patients who admitted to hospital within 72h after onset were randomized into treatment group (n=12) and control group (n=12). The patients in the treatment group were treated by Yihuo Qingxia method, and the control group were administrated with placebo.The level of the serum mcp1 of the patients on the first,3rd,5th,7thday were measured, as well as the incidence and the duration of disfunction of the organs were compared.〖WTHZ〗Results :There were no statistical significance in admission Rason scores, CT scores, Acute physiology and chronic health evaltionⅡscores(APACHEⅡscores)(Pgt;005). The level of the serum Monocyte chemoattractant protein1 of the treatment group was lower than that of the placebo group generally(Plt;005).At the 3rd day after onset,the serum mcp1 level of the control group was significantly higher than that of the treament group(Plt;005).The incidence of the control group of the intestin disfunction and hepatic inadequacy was obviously higher than those of the treatment group,and the duration of the former was longer than that of the latter,but with no satistical significance. Conclusion :Yihuo Qingxia method can effectively cut down the level of the serum mcp1 of severe pancreatitis patients.
Objective To investigate the relationship of pulmonary surfactant protein D( SP-D) with chronic obstructive pulmonary disease ( COPD) by measuring SP-D level in serum and lung tissue of rats with COPD.Methods The rat COPD model was established by passive smoking as well as intratracheal instillation of lipopolysaccharide ( LPS) . Thirty male SD rats were randomly divided into a control group, a LPS group, and a COPD group( n =10 in each group) . The pathologic changes of lung tissue and airway were observed under light microscope by HE staining. Emphysema changes were evaluated by mean linear intercept ( MLI) of lung and mean alveolar number ( MAN) . The level of SP-D in serum was measured by enzymelinked immunosorbent assay ( ELISA) . The expression of SP-D in lung tissue was detected by Western-blot and immunohistochemistry.Results The MLI obviously increased, and MAN obviously decreased in the COPD group compared with the control group ( Plt;0.05) . There was no significant difference in the MLI and MAN between the LPS group and the control group ( Pgt;0.05) . The serum SP-D level was ( 49.59 ±2.81) ng/mL and ( 53.21±4.17) ng/mL in the LPS group and the COPD group, which was significantly higher than that in the control group [ ( 42.14±2.52) ng/mL] ( Plt;0.05) . The expression of SP-D in lung tissue was 0.56±0.01 and 0.63±0.01 in the LPS group and the COPD group, which was also obviously ber than that in the control group ( 0.39 ±0.01) ( Plt;0.05) .Meanwhile the SP-D levels in serumand lung tissue were higher in the COPD group than those in the LPS group ( Plt;0.05) . The levels of SP-D between serum and lung tissue were positively correlated in all three groups ( r=0.93, 0.94 and 0.93, respectively, Plt;0.01) .Conclusion Both the SP-D level in serum and in lung tissue increase significantly in COPD rats and correlate well each other, which suggests that SP-D may serve as a biomarker of COPD.
Objective To investigate the clinical significance of the amplification of CyclinD1 gene and overexpression of its protein product in breast cancer. Methods Semi-quantitative PCR and immunohistochemical techniques were used to examine the amplification of CyclinD1 gene and overexpression of its protein product in breast cancer, mammary tissues adjacent to the tumor, mammary benign disease and normal mammary tissues. Results The results showed that amplification of CyclinD1 gene was observed in 14 out of 62 (22.6%) breast cancer, the overexpression of its protein product was 48.4% (30/62). Compared with other mammary tissues, these findings were significant. Amplification of CyclinD1 gene and overexpression of its protein were positively associated with histological grade, and there was a b correlation between CyclinD1 protein overexpression and estrogen receptor (progestogen receptor). Conclusion The degree of CyclinD1 protein overexpression is not quite the same as the frequency of the amplification of CyclinD1 gene. It suggests that the overexpresstion of CyclinD1 protein is also attributed to some other mechanisms. Estrogen might stimulate the expression of CyclinD1. The detection of CyclinD1 gene and its protein in breast cancer has some clinical significances.
Objective To study the effects of heat shock proteins (HSPs) in the course of hepatic ischemia reperfusion injury (HIRI), and analyze its mechanism. Methods The relationship between HSPs and HIRI was studied by reviewing literatures. Results HSPs was a kind of stress protein induced after cell was sitmulated by the stress. It could improve body′s tolerance to tough situation. Though hepatic ischemia reperfusion usually results in serious hepatic injury, at the same time it could induce can increase the production of HSPs that can protect liver from and lessen ischemia reperfusion injury. Conclusion HSPs can improve the tolerance to HIRI and lessen injury. In addition, HSPs is thought to be markers of HIRI, and can be used as a efficient indicator to test the level of hepatic injury and assess prognosis.
Objective To explore the vitamin K level in Chinese maintenance hemodialysis (MHD) patients. Methods MHD patients and healthy subjects from our outpatient clinic were enrolled from 1 to 30 in March 2016. Demographic data was collected. Fasting serum samples from all subjects were collected for biochemistry tests and the measurement of known vitamin K-dependent proteins, i.e. matrix Gla protein (MGP), osteocalcin (OC) and uncarboxylated osteocalcin (ucOC). We also quantified the fraction of ucOC of total OC (%ucOC). Differences of these parameters between the two groups were analyzed. Results We enrolled 70 MHD patients as a test group and 70 healthy subjects as a control group. There was no significant difference in MGP between MHD group and the control group [(4.1±2.2) vs. (4.4±1.0) pg/mL, P=0.441]. The value of %ucOC was significantly higher in the MHD group than that in the control group [(79.3±19.3)% vs. (51.9±13.0)%, P<0.001]. Conclusions Deficiency of vitamin K appears common in Chinese MHD patients. Besides pathological reasons, dietary habit may also contribute to this phenomenon.
ObjectiveTo explore the relationship of levels of serum interleukin-6 (IL-6), tumor necrosis factor-α(TNF-α) and C-reactive protein (CRP) with lung function in elderly patients with stable COPD and whose pulmonary function classification was levelⅡor above. MethodsSixty elderly patients with stable COPD and with the pulmonary function classification of levelⅡor above and 35 age-matched healthy subjects in the Gansu Provincial Hospital from November 2012 to March in 2014 were recruited in the study.Serum IL-6, TNF-αand CRP levels were detected by electro-chemiluminescence immunoassay (ECLI), enzyme-linked immunosorbent assay and immunoturbidimetric assay, respectively.And their relationships with lung function were explored by Spearman correlation analysis. ResultsThe levels of serum IL-6[(33.0±15.1) mg/L vs.(15.9±8.7) mg/L], TNF-α[(53.8±20.1) pg/mL vs.(22.2±8.0) pg/mL] and CRP[(8.7±3.9) mg/L vs.(5.8±2.3) mg/L] were significantly higher in the stable COPD patients than those in the healthy controls (P < 0.01).With the increase of COPD severity grade, the levels of serum IL-6, TNF-αand CRP increased gradually, and the lung function of FEV1%pred and FEV1/FVC decreased gradually (P < 0.05).The levels of serum IL-6, TNF-αand CRP were negatively correlated with lung function (P < 0.05). ConclusionsThere is airway inflammation in elderly patients with stable COPD.Airway inflammation may be the reason of the decline of pulmonary function in patients with stable COPD.