【作者】 汪国友；

【导师】 邓友章；

【作者基本信息】 成都中医药大学 ， 中医骨伤科学， 2013， 博士

【摘要】 目的：关节软骨损伤已经成为骨科临床的常见疾病。软骨损伤的修复一直是关节外科领域的难点及研究热点。本课题采用以“少阳主骨”为理论基础拟定的少阳生骨方干预体外关节软骨细胞培养和关节软骨损伤的动物模型,通过CCK-8法检测细胞增值、免疫组化法检测Ⅱ型胶原、RT-PCR法检测Ⅱ型胶原mRNA表达、放射免疫分析法(RIA)测定细胞因子IL-1β的变化,以及对软骨损伤后关节软骨组织形态和超微结构的改变,在细胞和分子水平的层面,探讨“少阳生骨方”治疗软骨损伤的可行性及可能机制,为应用中医药理论治疗软骨损伤提供有力的实验依据和开辟新的途径。为下一步继续研究少阳生骨方修复软骨损伤的机理和从分子生物学及细胞学水平进一步阐明该方的作用奠定基础。方法本实验分为四个部分第一部分：少阳生骨方含药血清对体外培养SD大鼠关节软骨细胞的影响获取1周龄SD大鼠关节软骨细胞进行单层细胞培养,取第三代传代软骨细胞,按1*105/ml的密度接种培养瓶中,分少阳生骨方(实验组)、盐酸氨基葡萄糖胶囊(对照组)及生理盐水(空白组)三组干预并获得含药血清,每组按0.6%、1.2%、2.4%的血清浓度加入培养基中干预72小时。干预72小时候后采用CCK-8法检测细胞增值、免疫组化法检测Ⅱ型胶原、RT-PCR法检测Ⅱ型胶原mRNA表达及细胞形态变化。第二部分：少阳生骨方含药血清对体外SD大鼠去分化关节软骨细胞的影响获取1周龄SD大鼠关节软骨细胞进行单层细胞培养,取第五代传代软骨细胞,按1*105/ml的密度接种培养瓶中,分少阳生骨方(实验组)、盐酸氨基葡萄糖胶囊(对照组)及生理盐水(空白组)三组干预并获得含药血清,每组按0.6%、1.2%、2.4%的血清浓度加入培养基中干预72小时,干预72小时候后采用CCK-8法检测细胞增值、免疫组化法检测Ⅱ型胶原、RT-PCR法检测Ⅱ型胶原mRNA表达及细胞形态变化。第三部分：少阳生骨方对SD大鼠在体软骨损伤后软骨组织形态和基质胶原表型表达的影响选用在相同条件下喂养的6～8周清洁级SD大鼠(泸州医学院动物实验中心提供)27只,雌雄不限,体重200±20g。按照组内均衡一致原则随机编为3组,即少阳生骨方组实验组(S组)、盐酸氨基葡萄糖胶囊组对照组(A组)和空白对照组(K组),每组9只。无菌条件下在股骨滑车正中用一直径为2.5mm克氏针钻孔,形成全厚软骨缺损,成为关节软骨损伤动物模型,于造模完成后的第二天,开始灌胃药物,S组少阳生骨方灌胃,A组盐酸氨基葡萄糖胶囊灌胃,K组生理盐水灌胃。连续灌胃4周,分别于灌胃后4、8、12周每组随机处死3只大鼠获取双侧软骨标本,肉眼观察关节软骨损伤处修复软骨情况,标本HE、甲苯胺蓝、Masson染色,光镜观察,按Mankin评分标准由进行评分,免疫组化染色检测Ⅰ、Ⅱ型胶原表达。第四部分：少阳生骨方对在体软骨损伤后关节液IL-1β水平的影响参照实验三步骤分别于术后4周、8周、12周三个点处死动物前,于双膝关节分别用1ml无菌生理盐水冲洗,获取关节液,采用放射免疫分析法(RIA)测定IL-1B水平,并绘制标准曲线。结果1.采用联合酶消化方法建立大鼠软骨细胞体外培养体系,经倒置显微镜下形态学观察及甲苯胺蓝染色鉴定,确认分离培养的细胞符合软骨细胞的生物学特征、形态学、组织化学染色显示软骨细胞培养传3代以内可以保持表型的稳定。对软骨细胞增殖的观察显示：少阳生骨方(SYSGF)0.6%、1.2%、2.4%含药浓度的血清均能促进体外正常软骨细胞增殖,但增殖的效应不随浓度的递增而增加,以1.2%的含药血清浓度促进正常培养关节软骨细胞增殖最为明显,且在48时促进增殖作用最佳。其增殖作用强于空白组,但不及2.4%的氨基葡萄糖胶囊组；免疫组织化学结果显示：三组标本的胞浆可见片状或团块状棕红色颗粒染色,此为Ⅱ型胶原表达。1.2%空白血清诱导的软骨细胞表达Ⅱ型胶原蛋白最弱。1.2%SYSGF组表达Ⅱ型胶原蛋白比1.2%空白血清组数目多,且可见细胞分裂相,呈显著差异(P<0.05); RT-PCR法不同浓度目的基因与内参基因光密度比值统计结果。显示实验组Ⅱ型胶原基因的表达与空白组相比,均有表达的增加。但是少阳生骨方组和盐酸氨基葡萄糖组相比其结果没有统计学差异。以上结果说明中药少阳生骨方能在一定程度上促进软骨细胞增殖,促进软细胞合成分泌Ⅱ型胶原蛋白。2.不同浓度的药物血清对第五代软骨细胞干预72小时后倒置显微镜观察：实验组细胞形态呈梭形、三角形、多形性,细胞胞浆较多,生长状态很好,细胞活性较好。CCK-8法测定用药48小时后少阳生骨方组与与空白组相比,有统计学差异(P<0.05)。而第一天和第三天数据有差异,但没有统计学意义。免疫组化法测定72小时候后Ⅱ型胶原染色表达阳性率检测结果,三组实验组与空白组相比,其Ⅱ型胶原表达阳性率均好于空白对照组。但是少阳生骨方组和盐酸氨基葡萄糖组差异不大,其差异性没有统计学意义。RT-PCR法半定量观察去分化软骨细胞Ⅱ型胶原mRNA表达显示,实验组Ⅱ型胶原基因的表达与空白组相比,均有表达的增加。但是少阳生骨方组和盐酸氨基葡萄糖组相比其结果没有统计学差异。3.少阳生骨方对SD大鼠在体软骨损伤后软骨组织形态和基质胶原表型表达的影响结果显示：大体观察和组织学观察均显示S、A组软骨缺损修复组织的数量及质量均优于K组,且S组的修复效果强于A组。对比空白组,少阳生骨方和盐酸氨基葡萄糖胶囊均可提高软骨损伤后修复组织中Ⅱ型胶原的表达(P<0.05),但两组比较无统计学意义(P>0.05)。4.少阳生骨方对在体软骨损伤后关节液IL-1β水平的影响结果显示：三组关节液中IL-1β水平检测,S组和A比较：第4、8、12周A组高于S组(P<0.05)；第4、8、12周K组高于S组(P<0.05)；第4、8、12周K组高于A组(P<0.05)。治疗前后自身比较：S组第8周、第12周与第4周相比无明显差异(P>0.05)；A组第8周、第12周均低于第4周(P<0.05)；K组第4周>第8周>第12周(P<0.05)。结论1.不同浓度少阳生骨方含药血清,均能促进SD大鼠体外第三代软骨细胞的增殖及Ⅱ型胶原表达,但并不随含药血清浓度的增加而增加,1.2%浓度促进其增殖表达能力最强,过高的浓度可能抑制其增殖、表达。2.不同浓度少阳生骨方含药血清,均能提高SD大鼠体外第五代软骨细胞Ⅱ型胶原及蛋白多糖表达及合成,维持软骨细胞表型稳定,但作用有限。3.少阳生骨方能促进SD大鼠在体软骨损伤后软骨的修复,且修复组织更接近透明软骨。4.少阳生骨方能明显降低SD大鼠在体软骨损伤后关节液的IL-1β水平,从而促进软骨细胞的修复。更多还原

【Abstract】 objective:Articular cartilage injury has become a common orthopaedic clinical disease, but its ability to self-repair is extremely limited. and so has been the difficulty in joint surgery field and research focus. This subject based on the theory of "shaoyang dominating bone"to apply ShaoYangShengGuFang Prescription intervening with the rat articular chondrocyte in vitro culture and animal models of cartilage injury in vivo. By CCK-8method to detect cell proliferation, Immunohistochemical method to detect Ⅱtype collagen, RT-PCR method to detect11type collagen mRNA expression, radiation immunity analysis (RIA) determination of cytokine changes of1L-1β. And the change of articular cartilage tissue morphological and ultrastructure, at the level of cell and molecular, to explore the the feasibility and the possible mechanism of the treatment of cartilage damage and provide strong experimental evidence for the application of Chinese medicine theory,open new avenues for the treatment of cartilage injury. Laying the foundation for the next step to continue to study the mechanism of the Prescription repair cartilage damage and elucidate the role of the Prescription from the the molecular Biology and Cytology level, provide strong evidence for clinical promotion.Methods:this experiment was divided into four parts.Part I:Ef ects of ShaoYangShengGuFang Prescription containing serum on the articular chondrocyte of SD rat in vitro culturechondrocytes were obtained and cultured in vitro from1weeks SD rat. The third passages were seeded in culture flask according to the density of1*102/ml. They were randomly divided into three groups: ShaoYanShengGuFang group(experimental group, S group), glucosamine hydrochloride capsule group (control group, A group) and normal saline group (blank group, K group). Each group was incubated with serum concentrations of0.6%,1.2%and2.4%for72hours; Subsequently, CCK-8method is used to detect cell proliferation, immunohistochemical method to detect II type collagen, RT-PCR method to detect Ⅱ type collagen mRNA expression and cell morphology changes.Part II:Ef ects of ShaoYangShengGuFang Prescription containing serum on the differentiation of SD rat articular chondrocyte in vitro culturechondrocytes were obtained and cultured in vitro from1weeks SD rat. The fifth passages was seeded in culture flask according to the density of1*105/ml. They were randomly divided into three groups: ShaoYanShengGuFang group (experimental group), glucosamine hydrochloride capsule group (control group) and normal saline group (blank group). Each group was incubated with serum concentrations of0.6%,1.2%and2.4%for72hours; Subsequently, CCK-8method is used to detect cell proliferation, immunohistochemical method to detect Ⅱ type collagen, RT-PCR method to detect Ⅱ type collagen mRNA expression and cell morphology changes.Part Ⅲ:Efects of ShaoYangShengGuFang Prescription on the cartilage injury of SD rat in vivoTo select27Qing-jie SD rats from6to8weeks under the same conditions (Provided by Animal Experimental Center of Luzhou Medical College), male or female, weight200±20g. randomly divided into three groups:ShaoYanShengGuFang group(S group), glucosamine hydrochloride capsule group(A group)and normal saline group(K group). In sterile conditions,drilled a hole in the middle of femoral trochlear by a hollow needle with a diameter of2.5mm. full thickness cartilage defect formation. The second day, began to gavage drugs4weeks. Respectively after4,8,12weeks, randomly executed three rats in each group to obtain dual lateral cartilage specimens. Macroscopic observation of articular cartilage, HE, toluidine blue and Masson staining, microscope observation and Mankin grading scale, immunohistochemical method to detect Ⅰ,Ⅱ type collagen expression. Part Ⅳ:Efects of ShaoYangShengGuFang Prescription on the level of IL-1β of SD rat in vivoReference to the experiment of Part III, after4,8,12weeks, to obtain joint fluid with1ml sterile saline flush In the double knee joint. By radiation immunity analysis (RIA) to detect changes of IL-1β, and draw the standard curveResults:1. Establish rat articular chondrocytes in vitro culture system with collagenase and trypsin digestion, and observe the biological characteristics of chondroeytes with inverted microscope, and identified by toluidine blue staining, And confirm the separation of cultured cells in cartilage cells of biological characteristics, morphology, histochemical staining showed cartilage cell culture can maintain the stability of phenotypic in three generations. Observation of the cartilage cell proliferation by CCK-8methods:The SYSGF groups:0.6%,1.2%,2.4%serum concentration can promote normal cartilage cell proliferation in vitro,1.2%serum concentration promotes normal articular cartilage cell proliferation is most obvious.And the48h, promoting proliferation effect best. its proliferation effect was more than the blank group, but less than2.4%serum concentration of glucosamine capsules group. The result of imunohistochemistry show as follows:All groups’specimens expressed The type II collagen, and the expression site was only in the cytoplasm, with red-brown particles, the expression of type II collagen of blank serum group was lower than the SYSGF group. there was remarkable statistical difference(P<0.05). RT-PCR method different concentrations with internal optical density ratio statistics, Showed the experimental group II type collagen gene expression compared with the blank group, has increased expression. But compared with A group has no statistical differences.2. The fifth passages were incubated with serum concentrations of0.6%,1.2%and2.4%for72hours, inverted microscope shows: Experimental group were fusiform, triangular, pleomorphic form, the cell cytoplasm was plentiful, chondrocytes activity was better. After48hours compared with the blank group, had statistical difference by CCK-8methods (P<0.05). and there are differences in the data between the first day with the third day, but no statistical significance. After72hours compared with the blank group, II collagen expression of experimental group were better than that of blank group. But there were no statistical significance between S group with A group. Semi-quantitative RT-PCR methods to observe type Ⅱ collagen mRNA expression, compared with the blank group, there were statistical significance.But compared with the A group, had no statistical significance. 3. cartilage morphology and stromal collagen phenotype expression results show:General observation and histological analysis showed the S group A cartilage defect repair tissue both quantity and quality is better than the K group and S group effect is stronger than A Group repair. compared with the blank group, S group and A group can improve the expression of type II collagen in cartilage tissue repair after injury (P<0.05), but the two groups is no statistical significance (P>0.05).4. Three sets of synovial fluid in comparison with the level of IL-1β detection, After4、8、12weeks, A group is higher than S group (P<0.05), K group is higher than S group (P<0.05). K group is higher than A group(P<0.05).Own comparison before and after treatment:S group have no differences(P>0.05); A group and K group, the eighth week and twelveth week are lower than the fourth week(P<0.05).Conclusion:1. Different serum concentrations of SYSGF could promote proliferation and type II collagen expression of the third-generation chondrocytes in vitro, but not increased with the increase of the serum concentrations,1.2%serum concentration showed strongest expression ability, but the more concentration the less proliferation and expression. 2. Different serum concentrations of SYSGF could improve type Ⅱ collagen and proteoglycan expression and synthesis of the fifth-generation chondrocytes in vitro, and stable chondrocyte phenotype, but their role is limited.3. Serum of SYSGF could promote cartilage repair of the SD rats in vivo, and closer to transparent cartilage repair tissue.4. Serum of SYSGF could significantly reduce the joint fluid levels of IL-1β of the SD rats in vivo, thus promotes the repair of cartilage cells更多还原