【作者】 龚建文；

【导师】 庄洪；

【作者基本信息】 广州中医药大学 ， 中医骨科学， 2011， 博士

【摘要】 [目的]本课题以“肾主骨生髓”为理论依据,在前期研究的基础上,从去卵巢大鼠骨髓中分离出间充质干细胞建立体外培养系,结合细胞形态学、分子生物学技术,观察中药骨康方含药血清定向诱导骨髓间充质干细胞成骨分化的能力,免疫组化、Western blot法测定成骨分化标志物的表达情况,运用RT-PCR技术检测测定成骨特异性转录因子Cbfal的表达,并进一步分析骨康含药血清诱导MSCs向成骨分化的可能信号机理,阐明P38信号通路机制在去卵巢大鼠(骨质疏松症病理状态模型)MSCs成骨分化中的重要作用。这将进一步阐明骨康在防治骨质量减少性疾病中的应用价值及其作用机理,为中医药调控干细胞治疗骨质疏松症的机理提供实验依据,为传统中医药走向世界提供实验数据。[方法]1.SPF级雌性Sprague-Dawley大鼠34只,其中10只用于制备骨康含药血清,14只切除SD大鼠双侧卵巢,造成大鼠去势,建立骨质疏松症模型,剩余为正常对照。2.由补骨脂,制淫羊藿,熟地,黄芪,菟丝子等十味中药组成的中药骨康方熬制中药汤剂,按人与大鼠体表面积比计算中药骨康方给药剂量,给予中药骨康方灌胃,于最后一次用药后2小时行心脏采血,制备含药血清。3.分别取4周龄SPF级正常SD大鼠10只和骨质疏松症模型大鼠12只,麻醉注射下,无菌条件下,分离出双股骨及胫骨,用D-Hank’s和DMEM液分别反复冲洗股骨和胫骨髓腔,提取和分离MSCs,以浓度为0.25%的胰蛋白酶(0.1ml/cm2)37℃条件下消化,以L-DMEM完全培养基,培养与传代MSCs,建立MSCs体外培养模型。4.实验分组：依次分为：No1：正常组对照；No2：正常组成骨诱导剂组(Control组+50μg/ml维生素C+10mmol/Lβ-甘油磷酸钠+10-8mol/L地塞米松)；No3：正常组骨康组(骨康含药血清)；No4：去势组对照；No5：去势成骨诱导剂组；No6：去势骨康组。5.以茜素红染色检测各组的钙化结节情况,确定细胞外基质的矿物化；以放免分析方法测定细胞培养液中的骨钙素(BGP)含量；以免疫组织化学方法,检测成骨标记物ALP、OPN的表达情况,以确定成骨分化能力。6.以Western blot技术,检测P38蛋白、ATF-2的表达情况；用Real-Time PCR技术检测成骨标记物Runx2、P38蛋白、ATF-2的表达情况。7.统计方法数据用SPSS13.0软件包进行统计学分析,各组间比较采用t检验进行统计学处理。概率p<0.05表示在统计学上差异具有显著性。图像用计算机图像分析系统进行条带灰度值的分析。[结果]1.用放免分析方法对培养7d、14d后的细胞进行骨钙素(BGP)测定,N04(去势大鼠组)7d、14d检测的骨钙素(BGP)分别为3.98ug/L,4.31 ug/L,NO1(正常对照组)中骨钙素(BGP)分别为4.67 ug/L,4.85 ug/L,去势大鼠组均较正常对照组有明显的减少,有显著差异；对培养7d、14d后的细胞进行茜素红染色检测,检测钙化结节,确定细胞外基质的矿物化,N04(去势大鼠组)7d、14d检测的钙化结节数分别为1.16个,2.01个,NO1(正常对照组)中钙化结节数分别为1.67个,2.52个,去势大鼠组均较正常对照组有明显的减少,有显著差异；7天后行成骨标记物ALP、OPN的免疫组织化学染色,检测成骨标记物ALP、OPN的表达情况,以确定成骨分化能力,结果显示去势大鼠的成骨分化标记物ALP、OPN的阳性细胞数减少。2.通过放免分析方法对培养7d、14d后的细胞进行骨钙素(BGP)测定,N06(去势骨康组)7d、14d检测的骨钙素(BGP)分别为6.12ug/L,6.66 ug/L,N05(去势成骨诱导组)中骨钙素(BGP)分别为5.97 ug/L,6.94 ug/L,N04(去势对照组)中骨钙素(BGP)分别为3.98 ug/L,4.31 ug/L,去势诱导组与去势骨康组均较去势对照组有明显的增加,有显著差异；对培养7d、14d后的细胞进行茜素红染色检测,检测钙化结节,确定细胞外基质的矿物化,N06(去势骨康组)7d、14d检测的钙化结节数分别为3.12个,5.11个,N05(去势成骨诱导组)中钙化结节数分别为4.01个,5.79个,N04(去势对照组)7d、14d检测的钙化结节数分别为1.16个,2.01个,去势诱导组与去势骨康组均较去势对照组有明显的增加,有显著差异；7天后行成骨标记物ALP、OPN的免疫组织化学染色,检测成骨标记物ALP、OPN的表达情况,以确定成骨分化能力,结果显示去势骨康组、去势成骨诱导组的成骨分化标记物ALP、OPN的阳性细胞数显著增加。RT-PCR检测Runx2表达量：去势骨康组、去势成骨诱导组的Runx2表达量较去势对照组有明显增加,有显著性差异。3. Western blot检测成骨标记物P38、ATF-2的表达情况,去势大鼠组较正常大鼠组P38表达低,而骨康含药血清能上调P38的表达。ATF-2是P38的下游分子,主要调控细胞的增殖与凋亡,本实验中其表达趋势与P38相同；RT-PCR定量分析发现去势骨康方组的P38、ATF-2的表达与去势对照组相比,有显著差异。结论1、通过大鼠骨质疏松症动物模型的建立,以及含药血清的制备,对MSCs的分离、纯化培养及骨向诱导,成功地建立了中药骨康方体外骨向分化生物学细胞模型,建立研究中药骨康方对骨髓间充质干细胞向成骨细胞分化这一动态过程的调控机制的实验模型。2、实验发现去势大鼠组不同时间钙化结节数减少、骨钙素含量下降,成骨分化标记物ALP、OPN的阳性细胞数减少,表明去势大鼠MSCs成骨分化能力下降,进一步证明了绝经后骨质疏松发病原因是由于体内成骨前体细胞向成骨分化及成脂肪分化失衡的结果。3、实验发现去势骨康组BGP的含量均较去势对照组有明显的增加,有显著差异；去势骨康组钙化结节数均较去势对照组有明显的增加,有显著差异；去势骨康组的成骨分化标记物ALP、OPN的阳性细胞数显著增加,有显著性差异；RT-PCR检测Runx2表达量：去势骨康组的Runx2表达量较去势对照组有明显增加,有显著性差异。由此表明中药骨康方通过上调Cbfal表达,促进去势大鼠MSCs向成骨分化。4、实验发现Western blot检测成骨标记物P38、ATF-2的表达情况,去势大鼠组较正常大鼠组P38表达低,而骨康含药血清能上调P38的表达。ATF-2是P38的下游分子,主要调控细胞的增殖与凋亡,本实验中其表达趋势与P38相同。RT-PCR定量分析发现去势骨康方组的P38、ATF-2的表达与去势对照组相比,有显著差异。由此证明中药骨康方诱导去势大鼠骨质疏松MSCs成骨分化主要是通过激活了P38信号传导通路,并通过介导Cbfal活性和表达这一关键点从而发挥调控作用。5、从以上结果可以推测中药骨康方诱导去势大鼠骨质疏松MSCs成骨分化的调控机制是：骨康方→激活P38信号传导通路→MAPK转入核内→上调成骨细胞特异性转录因子Cbfal→结合成骨细胞特异性顺式作用元件2 OSE2→激活碱性磷酸酶、Ⅰ型胶原、骨桥蛋白及骨钙素等的表达→生物学效应→诱导MSCs成骨分化。更多还原

【Abstract】 The topic of " kidney governs the bones and engenders marrow "as the theoretical basis, in previous studies, based on the bone marrow from ovariectomized rats were isolated mesenchymal stem cells were cultured in vitro systems, combined with cell morphology, molecular biology techniques, observation Chinese medicine GuKang contained serum directed differentiation of bone marrow mesenchymal stem cell osteogenic differentiation capacity, immunohistochemistry, Western blot determination of osteogenic differentiation markers expression, detected using RT-PCR determination of bone specific transcription factor Cbfal expression, and further analysis of serum containing bone health to induce osteogenic differentiation of MSCs may signal mechanism to clarify P38 signal pathway in ovariectomized rats (osteoporosis, pathologic state model) MSCs into osteoblasts important role in. This will further clarify the quality of bone health in the prevention of bone disease in the reduction of value and mechanism for the regulation of stem cells in medical treatment of osteoporosis and provide experimental basis for the mechanism for the provision of traditional Chinese medicine into the world of experimental dataMethods1. To get thirty-four SPF Sprague-Dawley rats, ten of which are for the preparation of pastille serum of Gukang. Fourteen of them are ovariectomized to build osteoporotic models. Others are the normal control group.2. By the psoralen, the system Epimedium, Rehmannia, Astragalus, dodder, etc. composed of ten Chinese herbs brewed GuKang decoction of Chinese medicine, by human and rat body surface area than the calculated dose of GuKang side to give medicine GuKang gavage, in the last 2 hours after administration cardiac blood, prepared the pastille serum.3. Were taken 4 weeks old SPF SD rats with normal levels 10 and osteoporosis 12 rats, under anesthesia injection, sterile conditions, the isolated femur and tibia double, with D-Hank’s solution were repeatedly washed and DMEM Femur and tibia bone marrow cavity, extraction and separation of MSCs, the concentration of 0.25% trypsin (0. 1ml/cm2) 37℃under the conditions of digestion, the L-DMEM complete medium, cultured and passaged MSCs, MSCs established in vitro model.4. Experimental group:No1:normal control group; No2:control osteogenic agent group(Control group+50μg/ml Vitamin C+10mmol/Lβ-glycerophosphate +10-8mol/L dexamethasone); No3:normal Gukang group (pastille serum of Gukang medicine); No4:ovariectomized control group; No5:ovariectomized osteogenic agent group; No6:ovariectomized Gukang group.5. ALIZARIN RED staining for calcification of nodules in each group, identifying mineralized extracellular matrix; determined by radioimmunoassay in the culture medium osteocalcin (BGP) content; to immunohistochemistry to detect bone formation markers ALP, OPN expression, to determine the osteogenic differentiation capacity.6. Western blot technique to detect P38 protein, ATF-2 expression; with Real-Time PCR Detection of osteogenic markers Runx2, P38 protein, ATF-2 expression.7. Statistical MethodsData were analyzed with SPSS13.0 package, was used to compare each group t test and x2 test was used for statistical analysis. Probability p<0.05 indicates statistically significant difference. Images by computer image analysis system strip gray value analysis.Results1. Radioimmunoassay method using to detect osteocalcin (BGP) of the cells after cultured 7d,14d determination, N04 (castrated rats group) 7d,14d detection of osteocalcin (BGP) were 3.98ug/L,4.31 ug/L, NO1 (control group) and osteocalcin (BGP) was 4.67 ug/L,4.85 ug/L, ovariectomized group than in the control group rats were significantly reduced, with significant differences; After cultured 7d,14d the cells ALIZARIN RED staining, detection of calcified nodules, to determine the extracellular matrix mineralization, N04 (castrated rats group) 7d,14d detection of calcified nodules was 1.16,2.01, NO1 (control group) in the number of calcified nodules were 1.67,2.52, ovariectomized group than in the control group rats significantly reduced, with significant differences; after 7 days the bone formation markers ALP, OPN immunohistochemical staining, detection of osteogenic markers ALP, OPN expression, to determine the osteogenic differentiation capacity of adult ovariectomized rats showed that bone differentiation marker ALP, OPN positive cells decreased.2. Analysis by radioimmunoassay in the cells after cultured 7d,14d to detect osteocalcin (BGP), N06 (OVX GuKang group) 7d,14d detection of osteocalcin (BGP) were 6.12ug/L,6.66 ug/L, N05 (castration bone induction group) osteocalcin (BGP) was 5.97 ug/L,6.94 ug/L, N04 (OVX control group) and osteocalcin (BGP) was 3.98 ug/L,4.31 ug/L, OVX induced GuKang group and castrated group compared with OVX control group increased significantly, with significant differences; ALIZARIN RED staining on the cells cultured after 7d,14d to detect Calcified nodules, to determine the extracellular matrix mineralization, N06 (OVX GuKang group) 7d,14d number of calcified nodules detected were 3.12,5.11, N05 (castration bone induction group) in the calcified nodules Respectively 4.01,5.79, N04 (OVX control group) 7d, 14d number of calcified nodules detected were 1.16,2.01, OVX induced GuKang group and the ovariectomized group than in the control group, castrated A significant increase, with significant differences; after 7 days the bone markers ALP, OPN immunohistochemical staining to detect osteogenic markers ALP, OPN expression, to determine the osteogenic differentiation capacity of GuKang showed that castration Group, ovariectomized group osteogenic osteogenic differentiation markers ALP, OPN positive cells were significantly increased. RT-PCR, expression of Runx2:castration GuKang group, castration Runx2 osteogenic induction group than the OVX control group, the expression of a significant increase, a significant difference.3. After the detection of the expression of osteogenic markers P38 and ATF-2 with Western blot, low expression of P38 normal rats, castrated rats group expressed slightly increased, but not obvious, but the pastille serum of Gukang can increase the expression of P38. ATF-2 is a downstream molecule P38, the main regulation of cell proliferation and apoptosis, this experiment the same as the change in trends and P38; RT-PCR quantitative analysis showed that castration GuKang group P38, ATF-2 expression OVX control group, there were significant differences.Conclusions1. Osteoporosis in the rat animal model, and the preparation of drug-containing serum, the separation of MSCs, pure culture and osteogenic induced differentiation of bone biology to establish in vitro cell model, build up an experimental model to study the control of mechanism Gukang on the dynamic process of bone marrow mesench-ymal stem cells’differentiation to osteoblast on the basis of building osteoporotic models.2. It is found in the experiment that the osteoblast differentiation of MSCs in the ovariectomized rats decline, which is embodied in the decrease of calcified nodules, BGP content, the numbers of positive cells in osteogenic markers ALP and OPN at different times.3. It is found in the experiment that the osteoblst differentiation culture fluid can successfully induce the differentiation of MSCs in ovariectomized rats, which may be accelerated by the Chinese medicine Gukang through transfering the expression of Cbfal.4. Gukang induced osteogenic differentiation of MSCs in ovariectomized rats may activate the p38 signaling pathway, mediated through the activity and expression of Cbfal on the key regulatory role to play.5. From the above results suggested the possible regulatory mechanism: Gukang→activate the P38 signaling pathway→MAPK moves into nucleus→transfer the osteoblast-specific transcription factor Cbfal→combine the osteoblast-specific cis-acting element 2 OSE2→activate alkaline phosphatase, Type I Collagen, osteopontin and BGP, etc.→biological effect→induce MSCs osteoblast differentiation.更多还原