移植间充质干细胞在胶原诱导性关节炎大鼠关节的分布和作用

目的 探讨移植人的异基因骨髓间充质干细胞(BM-MSCs)在类风湿关节炎(RA)动物模型胶原诱导性关节炎(CIA)大鼠炎症关节局部的转归以及对关节局部组织损伤的修复作用.方法 分别选取5只Wistar幼年大鼠和30只Wistar成年雌性大鼠,5只Wistar幼年大鼠用以提取BM-MSCs,30只Wistar成年雌性大鼠随机分为CIA造模A组、B组和正常C组各10只.BM-MSCs分离、传代培养后用5-溴脲嘧啶脱氧核苷(5-BrdU)进行标记.CIA大鼠成功造模后,采取尾静脉注射移植于A组和C组大鼠,移植细胞数为1.0×107/kg.移植后1-4周观察各组动物一般状况和关节局部肿胀度的变化;于移植4周后用关节组织病理切片局部免疫组织化学抗5-BrdU和骨保护素的方法观察BM-MSCs向受体关节局部的趋化和聚集以及对关节组织中骨保护素表达的影响.2组间比较采用t检验.结果 BM-MSCs移植后,受试动物关节肿胀开始消退[(200±350)μl],移植后2、3、4周时与未移植BM-MSCs的B组动物[(320±110)μl]之间比较差异有统计学意义(P＜0.05);在受者关节滑膜、血管内膜及软骨组织中均可见5-BrdU阳性细胞,滑膜组织处的平均灰度值CIA造模A组(85±9)低于正常C组(110±6),差异有统计学意义(P＜0.05);BM-MSCs移植后CIA造模A组关节滑膜中骨保护素阳性率增加,平均灰度值为54±4,明显低于未移植的CIA造模B组(77±6),差异有统计学意义(P＜0.05).结论 通过外周静脉移植的BM-MSCs可以特异性趋化和聚集到炎症关节局部,并可以减轻关节损害,而这种局部组织损伤修复作用可能是通过增加局部炎症关节组织中骨保护素的含量而发挥的.

Abstract：

Objective To study the distribution of allogenic bone marrow-derived mesenchymal stem cells (BM-MSCs) on joints of collagen-induced arthritis (CIA) rats and to investigate their repair effects on joint damages. Methods Five Wistar rats were used for extraction of mesenchymal stem cells and 30 adult female Wistar rats were divided into 3 groups: the CIA rats group A (n=10), CIA rats group B (n=10) and normal rats control group C (n=10). BM-MSCs of Wistar rats were isolated, cultured in vitro routinely and the fourth passages was taken for identification of specific surface antigens by flow cytometry, then the cells were labeled with 5-bromodeoxyuridine (5-BrdU) in vitro. The models of CIA rats were established. 5-BrdU labeled BM-MSCs (1.0×107 cells/kg) were imfused from through tail vein to CIA rats group A and control group C. During the first 4 weeks after BM-MSCs transplantation, changes of general condition and left hind paw swelling were examined. At the fourth week, immunohistochemical examination of 5 -BrdU and osteoprotegerin (OPG) were performed to investigate BM-MSCs aggregation around the knee joints. The contribution of BM -MSCs to repairing of joint damages was identified. Comparisons between groups were performed by t-test. Results After BM-MSCs transplantation, left hindpaw swelling of group A were relieved compared with group B (P＜0.05) and the mobility of the joints was significantly improved. At the fourth week, much more implanted cells (5-BrdU positive cells.) were detected in the damaged knee joints than those in normal knee joints. The average grey scale values on synovium of knee joints in the CIA group A (85±9) was significantly lower than that of the normal group C (110±6, P＜0.05). At the same time, OPG expression was increased in damaged knee joints. The average grey scale values on synovium of knee joints in CIA group A (54±4) was significantly lower than that of the CIA group B (77±6, P＜0.05). Conclusion The transplanted allogeneic bone marrow mesenchy-mal stem cells can migrate to sites of damaged tissue in arthritis. They can prevent tissue damage and repair the damaged joints tissue by increasing OPG expression. This study has provided some evidence for developing effective therapy for rheumatoid arthritis.     

Protective effect of RadixAstragali injection on immune organs of rats with obstructive jaundice and its mechanism

AIM： To observe the protective effect of RadixAstragali injection on immune organs （lymph nodes, spleen and thymus） of rats with obstructive jaundice （OJ） and its mechanism.
METHODS： SD rats were randomly divided into sham-operation group, model control group and Radix Astragali treatment group. On days 7, 14, 21 and 28 after operation, mortality rate of rats, pathological changes in immune organs, expression levels of Bax and nuclear factor （NF）-κB p65 proteins, apoptosis indexes and serum tumor necrosis factor （TNF）-α level in spleen and thymus were observed, respectively.
RESULTS： Compared to model control group, the number of dead OJ rats in Radix Astragali treatment group decreased （P 〉 0.05）. The TNF-α level （27.62 ± 12.61 vs 29.55± 18.02, 24.61 ± 9.09 vs 31.52± 10.95） on days 7 and 21, the pathological severity score for spleen [0.0 （0.0） vs 0.0 （2.0） on days 7 and 14 and for lymph nodes [0.0 （1.0） vs 1.0 （2.0）, 1.0 （0.0） vs 2.0 （1.0）] on days 21 and 28, the product staining intensity and positive rate of Bax protein in spleen [0.0 （0.0） vs 1.0 （2.0）, 0.0 （1.0） vs 2.0 （1.5） and thymus [0.0 （0.0） vs 1.0 （2.0）, 0.0 （1.0） vs 2.0 （1.5）] on days 14 and 28, the apoptotic indexes [0.0 （0.0） vs 0.0 （0.01）] in spleen and thymus [0.0 （0.0） vs 0.0 （0.01） on days 14 and 21 were significantly lower in Radix Astragali treatment group than in model control group （P 〈 0.05）.
CONCLUSION： Radix Astragali has protective effects on immune organs of OJ rats by relieving the pathological changes in immune organs, reducing TNF-α level and inhibiting Bax expression and apoptosis in spleen and thymus.     

血管内皮生长因子基因转染骨髓间充质干细胞移植对肺气肿大鼠的疗效

目的：观察血管内皮生长因子（VEGF）基因转染大鼠骨髓间充质干细胞（MSCs）移植对肺气肿大鼠肺泡壁细胞的修复作用。方法：pcDNA3．1-hVEGF转染雄性Lewis大鼠MSCs。雌性Lewis大鼠随机分为4组：正常对照组、肺气肿组、单纯MSCs移植组,hVEGF移植组。观察MSCs移植28d后肺组织形态学变化,支气管肺泡灌洗液细胞分类计数,ELISA法检测VEGF含量。Y染色体荧光原位杂交（Y-FISH）示踪雄性大鼠MSCs在雌性肺气肿大鼠肺组织内的植入情况;采用TUNEL法检测肺泡壁细胞凋亡,免疫组化法检测Bcl-2蛋白的表达。结果：hVEGF移植组肺气肿改变较肺气肿组、单纯MSCs移植组减轻;支气管肺泡灌洗液炎性细胞数较后2组减少;肺组织VEGF含量较后2组增多;肺泡壁细胞凋亡指数明显低于后2组;Bcl-2染色阳性细胞百分比高于后2组;Y-FISH显示hVEGF移植组、单纯MSCs移植组的雌性大鼠肺组织中有Y染色体阳性的细胞。结论：真核表达载体pcDNA3．1hVEGF可以成功地在MSCs中表达,pcDNA3．1hVEGF转染MSCs移植能够减轻大鼠肺气肿样改变,其疗效优于单纯MSCs移植治疗。

Abstract：

Objective： To evaluate the effect of mesenchymal stem cells （MSCs） transfected with human vascular endothelial growth factor （VEGF） gene for rats with pulmonary emphysema. Methods： MSCs from male lewis rats were transfected with the pcDNA3. 1-hVEGF control vector. Female Lewis rats were randomly divided into four groups： normal control group, emphysema group, emphysema ＋ MSCs transplantation group, emphysema ＋ hVEGF-transfected MSCs transplantation group. Morphologie changes of the lung tissue were observed 28 Jays after treatment. The total cell number of bronchoalveolar lavage fluid were counted, and the content of VEGF was de tected by ELISA. The engraftment of male bone marrow MSCs in female recipient lung was determined by Y chromosome fluorescent in situ hybridization （Y-FISH）. The apoptosis of the lung cells was assessed by TUNEL stai ning. The expression of Bcl-2 were determined by immunohistochemical staining. Results： Emphysematous change in the emphysema ＋ hVEGF transfeeted MSCs transplantation group was improved compared with those in emphysema group and the emphysema ＋ MSCs transplantation group. The total cell number of bronchoalveolar lavage fluid was decreased in the emphysema ＋ hVEGF transfected MSCs transplantation group compared to the emphysema group and the emphysema ＋ MSCs transplantation group. The level of VEGF was higher in emphysema ＋ hVEGF transfected MSCs transplantation group compared with those of the emphysema group and the emphysema ＋ MSCs transplantation group. The apoptotic index of the alveolar wall cells in the emphysema ＋ hVEGF transfeeted MSCs transplantation group was less than that of the emphysema group and the emphysema ＋ MSCs transplantation group. The percentage of Bcl-2 positive cells in the emphysema ＋ hVEGF transfected MSCs transplantation group was significantly higher than that of the emphysema group and the emphysema ＋ MSCs transplantation group. Y chromo some positive cells were observed in the lungs of rats from the emphysema ＋ hVEGF transfected MSCs transplantation group and the emphysema ＋ MSCs transplantation group, Conclusions： The hVEGF gene could expressed in MSCs. Trans plantation of MSCs transfected with hVEGF gene can improve emphysematous changes than single cellular therapy.     

Effects of electroacupuncture at “Zusanli” (ST 36)on expression of mitophagy-related proteins in skeletal muscle in rats with spleen deficiency syndrome

正Objective To observe the expression change of mitophagy-related proteins in skeletal muscle in rats with spleen deficiency syndrome and to explain the partial action mechanism of acupuncture at Zusanli (ST 36) for spleen deficiency syndrome.Methods Forty male SD rats, after normal feeding, were randomly divided into a normal group, a spleen deficiency group, a Zusanli group and a non-acupoint group, ten rats in each group.Except the normal group, the three factors modeling method was used for 14 days to establish the model of     

The regulatory impact of immune inhibitors on T cells of SD rats

Objective:To observe the regulatory impact of immune inhibitors on T cells in rats.Method:Forty SD rats were selected and randomly divided into experimental group and control group.Rapamycin(SRL)0.4 mg/d to fill the stomach of the former one,saline lavage was used with the latter one for two weeks.Using flow cytometry to detect the two groups of rats with spleen and thymus level of CD4+CD25+T cells;and the spleen cells FoxP3 mRNA expression;Using ELISA method to detect TGF-β,IL-10 levels.Results:The peripheral blood,spleen and thymus of CD4+CD25+T cells accounted for the proportion of mononuclear cells were significantly higher than that of control group(P0.05);FoxP3 mRNA expression quantity also significantly higher than the control group(P0.05);Experimental TGF-βin rats,IL-10 levels are significantly higher than control group(P0.05).Conclusions:Immune inhibitors can regulatory CD4+CD25+foxp3+T cells in rats,a single nuclear cell proportion increase,shows that it can induce rat CD4+CD25+foxp3+regulatory T cells proliferation.     

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白对炎性关节炎患者关节置换术后恢复的影响

目的 探讨重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc)对炎性关节炎患者关节置换术后恢复的影响.方法 回顾分析67例应用rhTNFR:Fc或传统改变病情抗风湿药(DMARDs)治疗的炎性关节炎患者行关节置换术后伤口感染发生例数、伤口愈合时间、炎症期时间(体温≥37.5 ℃)及抗生素应用时间.根据所应用药物分为rhTNFR:Fc组和传统DMARDs组.其中,rhTNFR:Fc组单用rhTNFR:Fc或rhTNFR:Fc联合传统DMARDs;传统DMARDs组单用或联合应用2种或2种以上传统DMARDs.统计学处理根据数据类型选择t检验或非参数检验.结果 67例患者中,rhTNFR:Fc组18例,传统DMARDs组49例.rhTNFR:Fc组1例出现伤口感染,传统DMARDs组0例,差异无统计学意义(P＞0.05).rhTNFR:Fc组炎症期时间为(4±3) d,传统DMARDs组为(3±3)d,差异无统计学意义(P＞0.05).rhTNFR:Fc组伤口愈合时间为(14.0±3.1)d,传统DMARDs组为(14.7±2.9)d,差异无统计学意义(P＞0.05).rhTNFR:Fc组术后抗生素应用时间为(14.8±9.3)d,传统DMARDs组为(10.3±2.7)d,差异有统计学意义(P＜0.05).结论 炎性关节炎患者围手术期应用rhTNFR:Fc不增加关节置换术后伤口感染发生率,不延长伤口愈合时间及炎症期时间.

Abstract：

Objective To investigate the affect of rhTNFR:Fc on the postoperative recovery of patients with inflammatory arthritis after arthroplasty. Methods Patients with inflammatory arthritis undergoing arthroplasty were included and divided into rhTNFR:Fc group (rhTNFR:Fc only or combined with conven-tional DMARDs) and conventional DMARDs group (monotherapy with or combination of conventional DMARDs). We retrospectively analyzed the incidence of postoperative infection, wound healing time, the febrile period (body temperature ≥37.5 ℃) and the duration of antibiotics treatment after arthroplasty. x2 test and t test were used for statistical analysis. Results Sixty-seven patients were included, 18 in the rhTNFR: Fc group and 49 in the conventional DMARDs group. One postoperative infection occurred in rhTNFR :Fc group but none in the DMARDs group. There was no significant difference by Fisher's exact test (P＞0.05). The febrile duration was (4±3) days in the rhTNFR :Fc group and (3±3) days in the conventional DMARDs group, the difference was not statistically significant (P＞0.05). The wound healing time was (14.0±3.1) days in the rhTNFR :Fc group and (14.7±2.9) days in the conventional DMARDs group, which was not statistically different(P＞0.05). The duration of antibiotics treatment after operation was (14.8±9.3) days in the rhTNFR: Fc group and (10.3±2.7) days in the conventional DMARDs group, the difference was statistically significant (P＜0.05). Conclusion Using rhTNFR:Fc during perioperative period in patients with inflammatory arthritis does not increase the risk of infectious complications or extending wound healing time and the febrile duration.     

Effect of operation-synchronizing transfusion of apoptotic spleen cells from donor rats on acute rejection of recipient rats after liver transplantation

AIM: To study effect of operation-synchronizing transfusion of apoptotic spleen cells from donor rats on acute rejection of recipient rats after liver transplantation. METHODS: Two of Wistar rats were chosen randomly for normal liver pathology control and ten of SD rats chosen randomly for liver function control as blank group (no operation). The rest of Wistar and SD rats were divided into four groups: control group (only liver transplantation), Dex group (donors receiving intraperitoneal injection of dexamethasone), SpC group (recipients receiving infusion of spleen cells of donors), Dex-SpC group (recipients receiving infusion of apoptotic spleen cells of donors), with each group except blank group, containing 10 SD rats and 10 Wistar rats, respectively. Wistar rats received liver transplantation from SD rats, in the meantime they received infusion of spleen cells of donors, which were induced by an intraperitoneal injection of dexamethasone (3 mg/(d.kg)·b.w) for three days before liver transplantation. The serum alanine transaminase (ALT), total bilirubin (T bili), liver pathological changes and survival time were analysed. Statistical analysis was carried out using SPSS 10.0 for Windows. Differences of the parametric data of ALT in means were examined by one-way ANOVA. Differences of ALT between two groups were examined by LSD. Differences of the nonparametric data of T bili in means and scores of pathology classification for acute rejection were examined by Kruskal-Willis H test. The correlations between ALT and T bili were analysed by Bivariate. Kaplan-Meier curves were used to demonstrate survival distribution. The log-rank test was used to compare the survival data. RESULTS: There were significant differences in ALT of the five groups (F= 23.164 P= 0.000), and ALT in Dex-SpC group was significantly higher than that in blank control, control, Dex, and SpC groups (P = 0.000), and ALT in SpC group was significantly higher than that in blank control (P= 0.000), control (P= 0.004), and Dex groups (P= 0.02). Results of nonparametric analysis of T bill showed that there were differences in T bill of the five groups (X2= 33.265 P= 0.000). T bili in Dex-SpC group was significantly higher than that in blank control, control, Dex, and SpC groups. T bili in SpC group was higher than that in blank control, control, and Dex groups. There were significant differences in scores of pathology classification for acute rejection in each of the groups (X2= 25.933, P= 0.000). The pathologically more serious acute rejection was found in Dex-SPC group than in other groups. No sign of acute rejection was observed in the blank control group. Slight acute rejection was observed in the control group. Slight-moderate acute rejection was observed in the Dex group. Moderate-acute rejection was observed in the SpC group. Severe-acute rejection was observed in the Dex-SpC group. The survival time in Dex-SpC group was shorter than in other groups (statistic = 11.13, P= 0.011). ALT and T bili were positively correlated (r= 0.747, P= 0.000, two-tailed). CONCLUSION: In order to reduce quantity of blood loss from rats after liver transplantation, only one of ALT or T bili is needed for liver function measurement of rats. Simultaneous injection of apoptotic spleen cells from donors induced by dexamethasone to liver transplantation rats aggravates acute rejection. One important mechanism of aggravation of acute rejection may be that apoptotic cells are not removed in time and that dead cells including apoptotic cells release inflammatory factors.     

重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白在胶原诱导性关节炎大鼠中的疗效观察及其对骨桥蛋白的影响

目的 观察重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc)在胶原诱导性关节炎(CIA)大鼠中对滑膜、关节软骨等的保护作用,并分析其对骨桥蛋白表达的影响.方法 建立CIA模型,第13天开始对治疗组采用rhTNFR:Fc腹腔注射治疗(10 mg/kg,隔日1次),每周测量体质量及踝关节的前后径,第36天处死大鼠,苏木素-伊红(HE)及甲苯胺蓝染色观察踝关节组织病理变化并进行病理评分;酶联免疫吸附试验(ELISA)检测血浆肿瘤坏死因子(TNF)-α和骨桥蛋白水平,免疫组织化学法检测踝关节骨桥蛋白的组织学表达.多组间均数比较采用单因素方差分析.结果 病理评分定量分析结果 显示造模组与治疗组比较,治疗组关节病理评分显著降低(分别为8.2±1.0与4.8±1.4,P<0.05);ELISA 结果 显示造模组平均血浆TNF-α和骨桥蛋白值分别为(713±146)pg/ml,(4.3±0.6)ng/ml,治疗组分别为(68±20)pg/ml,(4.2±0.6)ng/ml,2组间比较,血浆TNF-α值差异有统计学意义(P<0.05),而骨桥蛋白值差异无统计学意义(P=0.688);免疫组织化学显示骨桥蛋白主要表达在滑膜衬里层、软骨表面,造模组与治疗组间比较差异有统计学意义(P<0.05).结论 rhTNFR:Fc能显著减轻CIA大鼠关节及软骨的破坏,并能显著减少骨桥蛋白在关节滑膜的表达,延缓病情的进展.但不能减少骨桥蛋白在外周血浆中的表达,推测其原因可能为骨桥蛋白不直接参与炎症的发展过程,而主要参与骨质的破坏及吸收过程.

Abstract：

Objective To investigate the protection effects of recombinant human tumor necrosis factor-α receptor Ⅱ :IgG Fc fusion protein for injection (rhTNFR:Fc) on rats with collagen-induced arthritis (CIA) and analyze osteopontin (OPN) changes following therapy in order to understand its primary mechanism of action. Methods CIA was induced by bovine Ⅱ collagen (B Ⅱ C) injection. Rats were treated with rhTNFR:Fc from the 13th day after the first injection of B Ⅱ C till the 36th day. The anterior-posterior diameters of ankle joints and weight were measured weekly. The pathological score was evaluated by HE staining and toluidine blue staining. The blood plasma TNF-α and OPN levels were measured by ELISA and the histology expression was evaluated by immuno-histochemistry. Comparisons between groups were performed with one-way ANOVA. Results Quantitative analysis showed pathological score in the model group and treatment group was significantly reduced in joint pathology (8.2±1.0 vs 4.8±1.4, P<0.05). The mean plasma levels of TNF-α and OPN values were (713±146) pg/ml, (4.3±0.6) ng/ml respectively in the model group,but those of the treatment group were (68±20) pg/ml, (4.2±0.6) ng/ml. Serum TNF-α values were significantly different (P<0.05) between the two groups, while no significant difference was found in the value of plasma OPN (P=0.688) between the two groups. rhTNFR:Fc could reduce the cells OPN expression in the interface layer of the synovium and cartilage (P<0.05). Conclusion Pathology scores and ELISA results haveshown that rhTNFR:Fc has good therapeutic efficacy. It can significantly reduce the bone and cartilage damage of CIA mouse model, and can significantly reduce the expression of OPN in the sliding joints, thereby delay disease progression. However, it can not reduce the expression of OPN in the peripheral blood plasma.OPN may be involved in bone destruction and resorption rather than in inflammatory process.     

Intraportal mesenchymal stem cell transplantation prevents acute liver failure through promoting cell proliferation and inhibiting apoptosis

BACKGROUND: Transplantation of mesenchymal stem cells(MSCs) has been regarded as a potential treatment for acute liver failure(ALF), but the optimal route was unknown. The present study aimed to explore the most effective MSCs transplantation route in a swine ALF model.METHODS: The swine ALF model induced by intravenous injection of D-Gal was treated by the transplantation of swine MSCs through four routes including intraportal injection(In P group), hepatic intra-arterial injection(AH group), peripheral intravenous injection(PV group) and intrahepatic injection(IH group). The living conditions and survival time were recorded. Blood samples before and after MSCs transplantation were collected for the analysis of hepatic function. The histology of liver injury was interpreted and scored in terminal samples. Hepatic apoptosis was detected by TUNEL assay. Apoptosis and proliferation related protein expressions including cleaved caspase-3, survivin, AKT, phospho-AKT(Ser473), ERK and phospho-ERK(Tyr204) were analyzed by Western blotting.RESULTS: The average survival time of each group was 10.7 ±1.6 days(In P), 6.0±0.9 days(AH), 4.7±1.4 days(PV), 4.3±0.8 days(IH), respectively, when compared with the average survival time of 3.8±0.8 days in the D-Gal group. The survival rates between the In P group and D-Gal group revealed a statistically significant difference(P0.01). Pathological and biochemical analysis showed that liver damage was the worst in the D-Gal group, while less injury in the In P group. Histopathological scores revealed a significant decrease in the In P group(3.17±1.04, P0.01) and AH group(8.17±0.76, P0.05) as compared with that in the D-Gal group(11.50±1.32). The apoptosis rate in the In P group(25.0%±3.4%, P0.01) and AH group(40.5%±1.0%, P0.05) was lower than that in the D-Gal group(70.6%±8.5%). The expression of active caspase-3 was inhibited, while the expression of survivin, AKT, phosphoAKT(Ser473), ERK and phospho-ERK(Tyr204) was elevated in the In P group.CONCLUSIONS: Intraportal injection was superior to other pathways for MSC transplantation. Intraportal MSC transplantation could improve liver function, inhibit apoptosis and prolong the survival time of swine with ALF. The transplanted MSCs may participate in liver regeneration via promoting cell proliferation and suppressing apoptosis during the initial stage of ALF.     

帕金森病运动并发症发生机制中G蛋白偶联受体激酶6与N-甲基-D-天冬氨酸受体关系的研究

目的 研究G蛋白偶联受体激酶6(GRK6)在帕金森病(PD)运动并发症发生机制中与N-甲基-D-天冬氨酸(NMDA)受体的关系.方法 建立PD运动并发症大鼠模型,25只大鼠分为3组.异动症(LID)组10只,腹腔注射左旋多巴甲酯23 d;MK-801处理组10只,第23天左旋多巴甲酯注射前腹腔注射MK-801;PD组5只,腹腔注射0.2%维生素C液.另设假手术组5只为对照组.观察MK-801处理左旋多巴诱导的运动并发症模型大鼠的行为变化,并用免疫组织化学方法和Western印迹方法检测大鼠纹状体区GRK6蛋白的表达情况.结果 PD组大鼠长期使用左旋多巴后出现明显的异常不自主运动,与人类LID具有相似特征.免疫组化结果显示.PD组损伤侧纹状体区GRK6阳性细胞指数减少至(4.81±1.31)×103(P＜0.05),LID组损伤侧GRK6阳性细胞指数进一步减少至(3.23±0.41)×103(P＜0.01).MK-801组LID大鼠异常不自主运动评分减少,药效期延长,同时损伤侧纹状体区GRK6阳性细胞指数增多至(4.64±1.39)×103(P＜0.05).Western印迹法检测结果同免疫组化基本相符,PD组损毁侧/未损毁侧纹状体区GRK6含量比值为(83.7±2.1)%,LID组GRK6值降低为(76.8±2.2)%,而MK-801组GRK6值升高至(91.1±2.7)%(P＜0.01).结论 NMDA受体拮抗剂能逆转大鼠运动并发症的发生,其机制可能与GRK6增多,抑制了谷氨酸受体的过度活化有关.

Abstract：

Objective To investigate the relationship between G protein-coupled receptor kinase 6 (GRK6) and N-methyl-D-aspartate (NMDA) receptor in the mechanism study underlying motor complications in Parkinson's disease (PD).Methods The rat models (n= 25) of Parkinsonism related motor complications were established and were randomly divided into levodopa-induced dyskinesia (LID) group (n= 10,intraperitoneal injection of levodopa for 23 d),MK-801 treatment group (n= 10,intraperitoneal injection of MK-801 at day23 after intraperitoneal injection with levodopa for 22 days) and PD group (n= 5,intraperitoneal injection of vitamin C).Another 5 rats were served as controls (sham-operation group).The behavior changes of rats in MK-801 treatment group were observed,and the expression of GRK6 in the striate of rats was detected by immunohistochemistry and Western blot.Results After the chronic treatment with levodopa methyl ester,PD rats displayed abnormal involuntary movements,which was similar to levodopainduced dyskinesia in PD patients.Immunohistochemistry showed that GRK6-positive cells of lesion side were decreased in LID rats as compared with PD rats [(3.23±0.41 ) × 103 vs.(4.81 ± 1.31 ) ×103,P＜0.01].Rats in MK-801 treatment group displayed the decreased AIM scores and increased peak rotation,and the increased GRK6-positive cells of lesion side as compared with LID rats (P＜0.05).Western blot showed that the levels of GRK6 was 83.7% ±2.1% in PD group (presented as lesion side/unlesion side),76.8% ± 2.2% in LID group and 91.1% ± 2.7% in MK-801 treatment group (intergroups comparison:all P＜0.05).These results were in accordance with the results of immunohistochemistry.Conclusions Antagonist of NMDA receptor can be used to reduce the motor complications in rats.It may be due to increased GRK6 which inhibits the overactivation of glutamic acid receptors.     

异基因骨髓间充质干细胞移植对胶原诱导性关节炎大鼠的免疫调节作用

目的 通过对类风湿关节炎(RA)动物模型胶原诱导性关节炎(CIA)大鼠的实验研究,观察异基因骨髓间充质干细胞(MSCs)移植对早期、晚期CIA大鼠免疫细胞和分子的免疫学作用,并探讨其在体内发挥免疫调节作用的机制.方法 采用密度梯度离心结合贴壁培养法体外分离、培养大鼠MSCs,细胞表型鉴定;建立CIA大鼠模型;MSCs移植均采用尾静脉注射,移植后第42天全部处死动物取脾,通过实时定量-聚合酶链反应(RT-PCR)测定Foxp3 mRNA的表达水平,流式细胞术测定CD4+CD25+调节性T细胞的变化.采用单因素方差分析、LSD-t检验法进行统计学分析.结果 早期、晚期CIA对照组CD4+CD25+调节性T细胞(1.6±0.6,1.4±0.6)和Foxp3 mRNA(0.88±0.20,0.91±0.12)的表达水平低于健康组和治疗组,差异有统计学意义(P＜0.05),早期治疗组CD4+CD25+调节性T细胞(5.0±0.4)比晚期治疗组(3.9±0.4)有所升高,差异有统计学意义(P＜0.05).结论 异基因MSCs移植可通过上调CIA大鼠体内CD4+CD25+调节性T细胞水平,促进Foxp3 mRNA的表达而发挥其在体内的免疫调节作用,早期治疗组的疗效优于晚期治疗组.     

骨髓间充质干细胞关节腔注射治疗胶原诱导性关节炎的实验研究

目的 观察骨髓间充质干细胞(BMSCs)关节腔注射治疗胶原诱导性关节炎(CIA)的疗效.方法 体外扩增原代间充质干细胞至P2代.大鼠胶原二次免疫后第7天,将等量0.9％氯化钠注射液及P2代细胞分别注射至CIA不同侧足踝关节处.2,4,8,12周后观测关节炎指数和足爪关节X线分析及指数评估;12周处死动物进行组织学观察.采用配对t检验进行统计学分析.结果 在不同观察时间点间充质干细胞治疗组与对照组的关节炎指数为:(第2周)3.18±0.62与3.84±0.35,(第4周)3.45±0.28与4.06±0.38,(第8周)3.86:±0.23与6.75±0.36,(第12周)4.23±0.43与7.86±0.66;X线放射学指数为:(第2周)2.04:±0.21与2.72:±.0.15,(第4周)2.52±0.47与4.06±0.38,(第8周)3.56±0.29与4.35±0.36,(第12周)3.73±0.43与4.86±0.62;组织学评分为:(第12周)2.34±0.22与3.52±0.55.结论 骨髓间充质干细胞关节腔注射治疗可以抑制CIA大鼠的病情发展,有望成为一种新的临床治疗手段.     

雷公藤多苷对胶原诱导性关节炎大鼠骨桥蛋白及其受体αvβ3表达的影响

目的 观察雷公藤多苷(TWP)治疗后胶原诱导性关节炎(CIA)大鼠骨桥蛋白及其受体αvβ3表达的变化情况,探讨TWP治疗RA的可能机制.方法 建立CIA大鼠模型,将造模成功的大鼠随机分为模型组、TWP治疗组,4周后取材并分别用免疫组织化学染色及酶联免疫吸附试验(ELISA)法检测健康组、模型组和TWP治疗组的骨桥蛋白及其受体αvβ3在滑膜、关节和血清中的表达.统计学处理采用方差分析.结果 健康组、模型组、TWP治疗组的外周血骨桥蛋白的含量分别为(5.7±2.9)、(7.8±6.2)、(5.0±1.9) ng/ml,差异有统计学意义(F=6.74,P=0.016).3组滑膜和软骨中骨桥蛋白的含量(平均灰度值表示)分别为229±15,81±15,93±13和211±17,91±19,100±15,差异有统计学意义(F=52.48,P＜0.01;F=18.98,P＜0.01).滑膜和软骨中dvβ3蛋白的含量(平均灰度值表示)分别为235±16,91±16,131±14和198±10,99±15,113±14,总体差异有统计学意义(F=23.03,P=0.002; F=12.04,P=0.008).经SNK法组间两两比较模型组大鼠外周血、滑膜、软骨中骨桥蛋白及滑膜和软骨中其受体αvβ3的表达明显高于健康组;治疗组大鼠外周血、滑膜、软骨中骨桥蛋白及滑膜和软骨中其受体αvβ3的表达显著低于模型组.结论 TWP治疗RA滑膜炎和骨质破坏的分子机制可能与其降低骨桥蛋白及其受体αvβ3的表达有关.     

骨髓间充质干细胞移植治疗胶原诱导性关节炎的疗效和机制

目的 探讨骨髓间充质干细胞(MSCs)治疗胶原诱导型关节炎(CIA)疗效及可能治疗机制.方法 分离培养C57BL/6小鼠MSCs.健康对照组DBA-1小鼠3只(第0天和第21天尾静脉输注生理盐水0.2 m1)15只牛Ⅱ型胶原诱导的CIA小鼠随机分为CIA对照组(第0大和第21天尾静脉输注生理盐水0.2 ml)、预防组(第0天输注MSCs1×106细胞数/200 μl)和治疗组(第21天输注MSCsl×106细胞数/200 μl).另设健康对照组(不诱导CIA,第0天和第21天尾静脉输注生理盐水0.2 ml).疗效观察包括关节炎指数(AI)评分,关节病理分析,酶联免疫吸附试验(ELISA)测血清肿瘤坏死因子(TNF)-α,白细胞介素(IL)-1β水平,流式细胞术检测脾脏和淋巴结CD4+CD25+Foxp3+T淋巴细胞百分率等.结果 ①预防组和治疗组AI、关节病理评分明显下降(P<0.05);②健康对照组、预防组和治疗组血清TNF-α和IL-1β水平降低(P<0.05),与AI呈显著正相关(P<0.05),TNF-α与关节病理评分呈显著正相关(r=0.61,P<0.01);③治疗组小鼠脾脏和淋巴结中调节性T细胞明显升高(P<0.05).结论 MSCs治疗CIA有效,抑制TNF-α和IL-1β和上调调节性T细胞可能是MSCs治疗CIA有效的机制.     

99Tc-亚甲基二膦酸盐对胶原诱导性关节炎大鼠核因子-κB受体活化因子配体/骨保护素系统的影响

目的 探讨锝[99Tc]-亚甲基二膦酸盐(99Tc-MDP)对Ⅱ型胶原诱导性关节炎(CIA)大鼠核因子-κB受体活化因子配体(RANKL)/骨保护素系统的作用.方法 建立CIA大鼠关节炎模型,分为空白组、模型组、甲氨蝶呤组和99Tc-MDP组,观察膝关节病理变化并评分;酶联免疫吸附试验(ELISA)法检测血清RANKL、骨保护素浓度;Western blot法检测滑膜RANKL和骨保护素表达.采用重复测量的方差分析和单因素方差分析进行统计学处理.结果 甲氨蝶呤组血清RANKL/骨保护素比值[给药后1周(1.076±0.016)、给药后2周(1.140±0.005)、给药后3周(1.155±0.023)],关节病理评分[滑膜(1.8±0.5)、软骨(1.9±0.6)、骨(1.8±0.5)],滑膜RANKL/骨保护素比值(1.156±0.014)明显低于模型组(1.269±0.025、1.296±0.015、1.340±0.011),(2.9±0.4、2.6±0.5、2.6±0.5),(1.340±0.013)(P＜0.01);99Tc-MDP组(1.035±0.034、0.986±0.019、0.991±0.020),(1.5±0.5、1.4±0.5、1.2±0.5),(0.098±0.026)明显低于甲氨蝶呤组(P＜0.01).结论 99Tc-MDP可能通过降低RANKL/骨保护素比值起延缓关节破坏的作用,其起效时间明显早于甲氨蝶呤.     

单用重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白及联合甲氨蝶呤对胶原诱导性关节炎大鼠关节骨破坏影响的实验研究

目的 通过建立胶原诱导性关节炎(CIA)大鼠模型,评价单用重组人Ⅱ型肿瘤坏死因子受体-抗体融合蛋白(rhTNFR:Fc)及其联合甲氨蝶呤在抑制CIA大鼠关节骨破坏方面的作用及机制.方法 利用皮下注射牛Ⅱ型胶原诱导Wistar大鼠发病,建立CIA大鼠模型.将造模成功,炎症评分≥2分的CIA大鼠随机分为生理盐水组(0.4 ml/周,腹腔注射)、甲氨蝶呤治疗组(1 mg周,腹腔注射)、rhTN FR:Fc治疗组(0.8 mg,每周2次,腹腔注射)、甲氨蝶呤+rhTN FR:Fc治疗组(甲氨蝶呤1 mg/周+rhTNFR:Fc 0.8mg,每周2次,腹腔注射).治疗8周后,处死大鼠,取踝关节拍摄X线片,胫骨上段行微计算机断层扫描技术扫描和制作硬组织切片,观察各组踝关节骨破坏情况,评价胫骨上段骨小梁变化及骨量变化.统计学处理采用SNK-q检验.结果 治疗8周后,rhTN FR:Fc组,甲氨蝶呤+rhTNFR:Fc组骨小梁面积百分数[(29.1±0.3)％,(26.7±0.6)％]及骨小梁数量(4.4±0.5)/mm,( 4.0±0.6 )/mm]明显高于0.9％氯化钠注射液组和甲氨蝶呤组[(12.9±0.5)％,( 13.2±0.4)％与(2.0±0.3 )/mm,(2.2±0.2)/mm](P＜0.01);rhTNFR:Fc组、甲氨蝶呤+rhTNFR:Fc组骨小梁分离度明显小于0.9％氯化钠注射液组和甲氨蝶呤组(P＜0.01).结论 单用rhTNFR:Fc及联合甲氨蝶呤均具有明显抑制关节骨破坏的作用,且其抑制炎症关节周围骨量减少的作用与抑制局部骨小梁数量减少及骨小梁分离度的增大相关.     

腺相关病毒介导的人护骨素对炎症性关节破坏的保护作用

目的 探讨携带人护骨素(hOPG)的重组腺相关病毒(rAAV-hOPG)对胶原诱导性关节炎(CIA)大鼠关节破坏的影响.方法 经牛Ⅱ型胶原诱导建立大鼠关节炎模型,随机分成3组:CIA空白对照组,增强绿色荧光蛋白(EGFP)阴性对照组,OPG治疗组,同时设立健康对照组,共4组,每组10只,于首次免疫后第25天开始,分别予磷酸盐缓冲液(PBS)、PBS、rAAV-EGFP、rAAV-hOPG双膝关节腔内注射50μl/侧.比较4组大鼠掌跖厚度、关节炎指数、病理评分、放射学评分、骨破坏因子及炎症因子蛋白表达情况.结果 冰冻切片证实AAV能有效转导关节滑膜组织.酶联免疫吸附试验(ELISA)法测得OPG蛋白的表达水平升高93%(P＜0.05),基质金属蛋白酶(MMP)-3蛋白表达下降35%(P＜0.05),而白细胞介素(IL)-1β造模组间比较差异无统计学意义.放射学骨破坏Larsen评分下降30%.结论 rAAV-hOPG能有效转导关节组织,表达OPC蛋白,显著减轻炎性关节炎的骨质破坏,可能对关节软骨有一定保护作用,对关节炎症无明显影响.