少阳生骨方对SD大鼠体外软骨细胞培养及在体软骨损伤影响的实验研究
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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。
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 culture
chondrocytes 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 culture
chondrocytes 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 vivo
To 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 vivo
Reference 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 curve
Results:
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
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。
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 culture
chondrocytes 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 culture
chondrocytes 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 vivo
To 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 vivo
Reference 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 curve
Results:
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
引文
[1]Garstang SV, Stitik TP. Osteoarthritis:epidemiology, risk factors and pathophysiology. Am J Phys Med Rehabil 85(Suppl 11), S2, quiz S12,2006.
[2]Ashiku SK, Randolph MA, Vacanti CA, et al. Tissue engineered cartilage[J]. Materials Science Forum,1997,2(50):129-150.
[3]Ahem BJ, Parvizi J, Boston R, et al. Preclinical animal models in single site cartilage defect testing:a systematic review. Osteoarthritis Cartilage,2009,17(6):705-713.
[4]Ibarra-Ponce de Kon JC, Cabrales-Pontigo M, Crisostomo-Martinez JF, et al. Results of arthroscopic debridement and lavage in patients with- knee osteoarthritis. Acta Othop Mex,2009,23(2):85-89.
[5]Fansa AM, Murawski CD, Imhauser CW, et al. Autologous osteochondral transplantation of the talus partially restores contact mechanics of the ankle joint. Am J Sports Med,2011,39(11):2457-2465.
[6]Yokota M, Yasuda K, Kitamura N, et al. Spontaneous hyaline cartilage regeneration can be induced in an osteochondral defect created in the femoral condyle using a novel double-network hydrogel. BMC Musculoskelet Disord,2011,12:49.
[7]Huang GY, Zhou LH, Zhang QC, et al. Microfluidic hydrogels for tissue engineering. Biofabrication,2011,3(1):5087-5092.
[8]MARLOVITS S, ZELER P, SINGER P C. et al. Cartilage repair:generations of autologous chondrocyte transplantation [J].Eur J Radiol,2006,57 (1):24-31.
[9]KANG SW, YOO SP, KIM BS, et al. Effect of chondrocyte passage number on histological aspects of tissue engineered cartilage [J].Biomed Mater Eng,2007,17(5):269-276.
[10]Kulkarni AB. Transforming growth factor B1 null mutation in mice causes excessive inflammtory response an dearly death. Proc Nat Ⅰ, Acad Sci USA,1993,90:770.
[11]郭向飞,胡声宇,徐亮等。中药“软骨康”对大鼠关节软骨Ⅰ型胶原、Ⅱ型胶原损伤修复的实验研究[J]。中国中医基础医学杂志,2008,14(8)：637-638。
[12]周尊谦,谢林,孙达武。丹紫康膝冲剂治疗膝关节退行性骨关节病临床研究[J]。中国骨伤,1999,12(2)：49-51。
[13]吕红斌,胡建中,罗承耀等。川芎嗪关节腔内注射对膝骨关节炎的治疗作用[J]。中南大学学报(医学版),2006,31(4)：591-594。
[14]刘舟。从《伤寒论》中的“支节颇疼”进而探讨《内经》中的“少阳主骨”[J]。光明中医杂志,2004,19(6)：2。
[15]谢健。足少阳主骨所生病在针灸临床的应用[J]。云南中医杂志,1994(06)：25-30。
[16]刘永臣。由柴胡桂技汤治“支节烦疼”体会“少阳主骨”[J]。中国中医药报,2005,4(4)。
[17]徐叔云。药理学实验方法[M]。北京：人民卫生出版社,2002,203-204。
[18]Manning WK, Bonner WM Jr.Isolation and culture of chondrocytes fromhuman articular cartilage [J]. Arthritis Rheum,1967,10(1):235-239.
[19]李元,杨庆铭,徐建强等。关节软骨细胞分离—胶原酶的细胞毒性作用初探[J]。上海医学,1998；21(2)：81-83。
[20]郝鹏,李胜富,裴福兴等。成年兔关节软骨创伤模型中软骨细胞的分离及体外培养观察[J]。华西医学,2007；22(1)：108-110。
[21]李昊。不同年龄兔软骨细胞培养和形态学研究[J]。山西职工医学院学报,2008：18(1)：15-17。
[22]Kurz B, Domm C, Jin M, et al. Tissue engineering of articular cart ilageunder the influence of collagen I/III membranes and low oxygen tens ion[J]. Tissue Eng,2004;10 (78):1277.
[23]Kart sogiannis V, Ng KW. Cell lines and primary cell cultures in the study of bone cell biology[J]. Mol Ceil Endoerinol,2004; 228(122): 79-82.
[24]夏万尧,曹谊林,商庆新等。组织工程化软骨组织形成的最佳细胞浓度和最佳形成时间的实验研究[J]。中国修复重建外科杂志,1999,13(4)：244-248。
[25]夏青,王卫东,魏振等。兔关节软骨细胞的体外培养及鉴定[J]。中国医师杂志,2007：9(9)：1174-1177。
[26]Dading EM, Athanasiou KA. Rapid phenotypic changes in passaged articular chondrocyte subpoulations[J]. Ortho Res,2005; 23(2):425-432.
[27]Schulze-Tanzil G, de Souza P, Villegas Castrejon H, et al. Rediffrentiation of dedifferentiated human chondrocytes in high-density cultures. Cell Tissue Res,2002; 308(3):371-379.
[28]Goessler UR, Bugert P, Bieback K, et al. Expression of collagen and tiber-associated proteins in human septal cartilage during in vitro dedifferentiation. Int J Mol Med,2004; 14(6):1015-1022.
[29]张群豪,陈可冀。血清药理学在中药及复方研究中应用的评价[J]。中国中西医结合杂志,1996；16(3)：131-133。
[30]杨奎,张德波,史焱等。含黄芩血清及黄芩甙影响内生致热产生的研究[J]。中药药理与临床,1994；13(6)：1410-1413。
[31冯伟。MTT法分析中药含药血清对体外软骨细胞增殖影响的研究[J]。上海中医药大学学报,2000；14(1)：43-45。
[32]崔晓兰,贺玉琢,肖英杰等。中药药理研究的新思路。中国中医药科技,1997；4(4)：239-241。
[33]冯伟,石印玉,沈培芝等。补肾方和补肝方含药血清对软骨细胞增殖动力学研究[J]。浙江中医学院学报,2001,25(4)：15-17。
[34]张荣华,舒晓春。中药复方补肾活血液对成骨细胞影响的实验研究[J]中国病理生理杂志,2003,19(6)769-772。
[35]许勇,杜宁,符诗聪等。健骨颗粒含药血清对人成骨细胞增殖芳香化酶活性的影响[J]。中国骨肿瘤骨病,2003,2(1)：51-54。
[36]刘明怀。黄芪注射液对病毒性心肌炎TNF-A、IL-1和IL-6的影响[J]。实用中医药杂志,2006,22(6)329-330。
[37]刘平,王宁生,雷燕等。关于血清药理学的若干思考[J]。中国中西医结 合杂志,1999：10(5)：263-266。
[38]秦梦,王和鸣,娄玉钤。不同浓度骨痹舒片含药血清对体外培养软骨细胞增殖的影响[J]。中国骨伤,2011,24(10)841-844。
[39]吕红斌,岳珍,王嘉芙。4种中药对体外培养兔关节软骨细胞代谢的影响[J]。中国运动医学杂志杂志,1995；14(3)：135-137。
[40]Kim SJ, Ju Jw, Oh CD, Yoon YM, Song WK, et al. TRK-1/2 and p38 kinase oppositely regulate nitric oxide-induced apoptosis of chondrocytes in association with p53 caspase-3 and differentiation status[J]. J Biol Chem,2002,277(2):1332-1339.
[41]Osaki M, Tan L, Choy BK, et al. The TATA-containing core promoter of the type II collagen gene(COL2A1) is the target of interferon gammamediated inhibition in human chondrocytes requirement for Statlalpha, Jakl and Jak2[J]. Biochem J,2003; 369(81):103-115.
[42]孙明林,朱雷,吕丹。Ⅱ型胶原蛋白对兔去分化软骨细胞再分化的作用[J]。中国修复重建外科杂志,2010,24(10)：1244-1247。
[43]刘瑾春,刘霞,曹谊林。软骨细胞培养中细胞去分化和再分化研究进展。中华整形外科杂志,2011,7,27(4)：318-320。
[44]Grigolo B, Lisignoli G,Piacentini A,etc. Evidence for redifferentiation of human chondrocytes grown on a hyaluronan-based biomaterial(HYAff 11):molecular, immunohistochemical and ultra-structural analysis Biomaterials.2002,23(4):1187-1195.
[45]Mukaida T, Urabe K, Naruse K, etc. Influence of three dimensional culture in a type II collagen sponge on primary cultured and dedifferen-tiated chondrocytes. Journal of Orthopaedic Science.2005,10(5):52.
[46]王卫国,娄思权,佘华等。TGF-β2转染关节软骨细胞的实验研究.中华骨科杂志,2003,7,23(7)：434-438。
[47]Kaps C, Bramlage C, Smolian H, etal. Bone morphogenetic proteins promote cartilage differentiation and protect engineered artificial cartilage from fibroblast invasion and destruction. Arthritis Rheum.2002,46(1):149-162.
[48]Messner K, Maletius W. The long-term prognosis for severe damage to weight-bearing cartilage in the knee:a 14 year clinical and radiographic follow-up in 28 young athletes. Acta Orthop Scand 67,165, 1996.
[49]Gotterbarm T, Breusch SJ, Schneider U, etal. The minipig model for experimental chondral and osteochondral defect repair in tissue engineering:retrospective analysis of 180 defects. Lab Anim 42,71, 2008.
[50]Wei X, Messner K. Maturation dependent durability of spontaneous cartilage repair in rabbit knee joint[J]. Jbiomed Mater Res,2004,46: 539-548.
[51]陈志群。针药并用治疗膝关节骨性关节炎50例临床观察[J]。中医药导报,2010,16(12)：72—73。
[52]Breinan HA, Mins TH, et al. Effect of cultured autologous chondrocytes on repair of chondral defect s in a canine model. J Bone Joint Surg (Am),1997,97:1439-1451.
[53]Gross RE. Repair of cartilage defects in the knee[J]. J Knee Surg, 2002,15(3):167-169.
[54]Buckw alt er JA, Rosenberg L, Cout ts R, etal. Articular cartilage: Injury and repair. Injury and Repair of the Musculoskeletal Soft Tissues 1988,465-482.
[55]胡炯,杜宁,代岭辉等。建立膝骨性关节炎关节软骨缺损的动物模型的研究[J]。中国中医骨伤科杂志,2009,17(11)：13-16。
[56]Mankin HJ, Mow VC, Buckwalter JA, et al. Format ion and function of arthcular cartilage. In:Sheldon R, Simon MD, eds. Orthopaedic BasiC Science. Rosemont, IL, American Academy of Orthopaedic Surgeons,1994, pp447-486.
[57]Allan DA. Structure and physiology of joints and their relationship to repetitive strain injuries. Clinc Orthop 351:32-38,1998.
[58]Tripple SB. Articular cartilage research. Curr Opin Rheumatol 2: 777-782,1990.
[59]Eyre D, Wu J. Collagens structure and cartilage matrixin tegrity. J Rheumatol,1995,22:82-85.
[60]Mandelbaum BR, Browne JE, Fu F, et al. Articular cartilage lesions of the knee。 Am J of Spor Med,1998,26(6):853-861.
[61]Heard F, Heard A,Harmand MF. Apoptosis in normal and osteoarthritic human articular cartilage[J]. Ann Rheum DiS,2000,59(12):959-965.
[62]Huber M, Trattnig S, Linner F. Invest Radiol,2000,35(10):573-580.
[63]Dodds RA, Merru K, littlewood A, et al. Expression of mRNA for IL-lb, IL1-6 and TGF b 1 in developing human bone and cartilage. J Histoehem Cytoehem,1994,42:733-744.
[64]Yamashita F, Sakakida K, Kusuzaki K, et al. Immunohistoehermical localization of interleukin-1 in human growth cartilage. Nippon Seikeigeka Gakkai Zasshi,1989,63:562-568.
[65]Dodds RA, Merry K, Littlewood A, et al. Expression of mRNA for IL-1 β, IL-6 and TGF B1 in developing human bone and cartilage. J Histochem Cytochem,1994,42:733-744.
[66]Teshima S, Nakanishi H, Nishizawa M, et al. Up-regulation of IL-lreceptor through PI3K/Akt is essential for the induction of iNOS gene expression in hepatocytes [J]. Hepatology,2004,40 (4):616-623.
[67]Kolettas E. Muir HI, Barrett JC, et al. Chondrocyte phenotype and cellsurvival are regulated by culture conditions and by specific Cytokines through the expression of sox-9 transcription factor. Rheumatology (oxford),2001,40:1146-1156.
[68]高洁生。骨关节炎治疗新视角[J]。中华风湿病学杂志,2008,12(12)：801-802。
1. Losina E, Walensky RP, Kessler CL, et al. Cost effectiveness of total knee arthroplasty in the United States:patient risk and hospital volume [J].Arch Intern Med.2009 Jun 22;169(12):1113-1121.
2. Ritzker KPH, Gay S, Jimenez SA. Osteoarthritis cartilage histo-pathology:grading and staging[J]. Osteoarthritis Cartilage,2006, 14:13-29.
3.袁淑娟,梁英,薛燕萍等。感觉运动训练治疗膝关节骨性关节炎的疗效观察。中华物理医学与康复杂志,2011,4；33(4)290-293。
4.赵焱全,加味四妙散为主治疗膝关节慢性滑膜炎体会[J]。中国现代药物应用,2009,3(18)：105—106。
5.金军,中药热敷治疗膝关节骨性关节炎180例临床观察[J]。中国中医基础医学杂志,2010,16(4)：344—346。
6.李俐,吴岩,何广富.血府逐瘀汤加减治疗骨性关节炎53例[J]。长春中医药大学学报,2007,23(6)：47。
7.何庆勇,张吉。针刺配合中药内服治疗膝骨性关节炎的临床观察[J]。时珍国医国药,2007,5(1)：36。
8.张智芳、王海明。温针灸治疗膝关节骨性关节炎的疗效观察。国际中医中药杂志,2010,32(3)：243-244。
9.王思茹,郭新惠,刘雪庆,发泡疗法治疗膝关节骨性关节炎的效果观察。国际护理学杂志,2011,29(7)1101-1103。
10.周兰芳,陈勇嫦,莫少媛,中药熏洗配合手法按摩治疗膝骨性关节炎的临床护理观察,国际医药卫生导报,2011,17(19)：2427-2429。
11. Haflah NH, Jaarin K, Abdullah S, et al. Palm vitamin E and glucosamine sulphate in the treatment of osteoarthritis of the knee. Saudi Med J,2009,30(11):1432-1438.
12. Black C, Clar C, Henderson R, et al. The clinical effectiveness of glucosamine and chondroitin supplements in slowing or arresting progression of osteoarthritis of the knee:a systematic review and economic evaluation. Health Technol Assess,2009,13(52):1.148.
13.江相保,高李侠。盐酸氨基葡萄糖治疗膝退行性骨性关节炎的临床观察。中国修复重建外科杂志,2008,22(1)：29-31。
14. Hempfling H. Intr-articulal hyalaronic acid after knee Arthro-scopy:a two-year study. Knee Surg Sports Traumatol Arthrosc,2007, 15:537-546.
15. Diracoglu D, Vural M, Baskent A, et al. The effect of viscosupple-mentation on neuromuscular control of the knee in patients with osteoarthritis. J Back Musculoskelet Rehabil,2009,22:1-9.
16.杨听,曹永平,刘超,文立成等。二膦酸盐对佐剂性关节炎的影响。中国实验外科杂志,2008,25(6)
17. Van Offel JF,Dombrecht EJ, Bridts CH, et al. Influence of bisphosphonates on the production of pro—inflammatory cytokines by activated human articular chondrocytes. Cytokine.2005,31(4): 298-304.
18. Hayami T, Pickarski M, Wesolowski GA, et al. The role of subchondral bone remodeling in osteoarthritis:reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model. Arthritis Rheum,2004,50(4):1193-1206.
19. Ding M, Danielsen CC, Hvid I. The effects of bone remodeling inhibition by alendronate on three dimensional micro arthitecture of subchondral bone tissues in guinea pig primary osteoarthrosis [J].Calcif Tissue Int.2008,82(1):77-86.
20.王予彬,王惠芳,朱文辉等。膝关节骨性关节炎关节镜下清理和联合髌外侧减压及康复治疗临床疗效的对比研究。中国微创外科杂志,2008,8：678-682。
21.潘惠娟,王予彬,王惠芳,卢亮宇。关节镜下清理髌外侧减压术配合康复训练治疗膝骨性关节炎的疗效观察。中华物理医学与康复杂志,2011,33(2),131-133。
22.余正红,蔡胥等。 膝关节神经分布的解剖学研究及其临床意义[J]。中国临床解剖学杂志,2008,26(1)：11-16。
23.肖春苟。兔膝关节神经支选择性切断后对关节软骨的影响[J]。解剖学研究杂志,2010,4(N)：13-15。
24. Pillings A, Scott DF, Camargo MP, et al. J Bone Joint surg Am, 2000; 82(1):70-79.
25. Zhim F. Laflamme G Y,Viens H, et al. Clin Biomech (Bristol Avon), 2005; 20 (8):871-876.
26. Trieb K, Grohs J, HansLik-Schnabel B, et al. Knee Surg Sports Traumatol Arthrosc,2006; 14(2):149-152.
27. Paccola CA, Fogagnolo E knee surg Sports Traumatol Arthrosc.2005,13(1):19-22.
28. Koshino T, Yoshida T。 Ara Y. et al. Fifteen to twenty-eight years' follow-up results of high tibial valgus osteotomy for osteoarthritic knee, Knee,2004,11(6):439-444.
29. Tang WC, Henderson IJ. High tibia] osteotomy: long term survival analysis and patients perspective. Knee,2005,12(6):410-413.
30. Rodriguez JA, Bhende H, Ranawat CS. Total condylar knee replacement: a 20-year Follow-up study. Clin Orthop Relat Res 2001,388:10-17.
31.Riddle DL, Jiranek WA。 McGlynn FJ. Yearly incidence of unicompartmental knee arthroplasty in the United States. J Arthroplasty,2008,23:408-412.
32. Jamali AA, Scott RD, Rubash HE, et al. Unicompartmental knee arthroplasty:past, present, and future. Am J Orthop,2009,38:17-23.
33.张启栋,郭万首,刘朝晖等.胫骨高位截骨与单髁关节置换治疗膝关节单问室骨关节炎的荟萃分析.中华医学杂志,2009,10,89(39)2768-2772。
34.刘朝晖,郭万首,张启栋等.单髁置换与全膝关节置换治疗膝关节单室骨性关节炎为主的早中期疗效比较,中华医学杂志,2010,10,90(37)：2597-2600。
35. Otten R. Van Roermund PM, Picavet HS. Trends in the number of knee and hip arthroplasties:considerably igore knee and hip prostheses due to ostoarthritis in 2030. Ned Tijdschr Geneeskd, 2010,154:A1534.
36. Wright JG, Santaguida PL, Young N. et al. Patient preferences before and after total knee arthroplasty. J Clin Epidemiol,2010,63 (7):774-782.
[2]Ashiku SK, Randolph MA, Vacanti CA, et al. Tissue engineered cartilage[J]. Materials Science Forum,1997,2(50):129-150.
[3]Ahem BJ, Parvizi J, Boston R, et al. Preclinical animal models in single site cartilage defect testing:a systematic review. Osteoarthritis Cartilage,2009,17(6):705-713.
[4]Ibarra-Ponce de Kon JC, Cabrales-Pontigo M, Crisostomo-Martinez JF, et al. Results of arthroscopic debridement and lavage in patients with- knee osteoarthritis. Acta Othop Mex,2009,23(2):85-89.
[5]Fansa AM, Murawski CD, Imhauser CW, et al. Autologous osteochondral transplantation of the talus partially restores contact mechanics of the ankle joint. Am J Sports Med,2011,39(11):2457-2465.
[6]Yokota M, Yasuda K, Kitamura N, et al. Spontaneous hyaline cartilage regeneration can be induced in an osteochondral defect created in the femoral condyle using a novel double-network hydrogel. BMC Musculoskelet Disord,2011,12:49.
[7]Huang GY, Zhou LH, Zhang QC, et al. Microfluidic hydrogels for tissue engineering. Biofabrication,2011,3(1):5087-5092.
[8]MARLOVITS S, ZELER P, SINGER P C. et al. Cartilage repair:generations of autologous chondrocyte transplantation [J].Eur J Radiol,2006,57 (1):24-31.
[9]KANG SW, YOO SP, KIM BS, et al. Effect of chondrocyte passage number on histological aspects of tissue engineered cartilage [J].Biomed Mater Eng,2007,17(5):269-276.
[10]Kulkarni AB. Transforming growth factor B1 null mutation in mice causes excessive inflammtory response an dearly death. Proc Nat Ⅰ, Acad Sci USA,1993,90:770.
[11]郭向飞,胡声宇,徐亮等。中药“软骨康”对大鼠关节软骨Ⅰ型胶原、Ⅱ型胶原损伤修复的实验研究[J]。中国中医基础医学杂志,2008,14(8)：637-638。
[12]周尊谦,谢林,孙达武。丹紫康膝冲剂治疗膝关节退行性骨关节病临床研究[J]。中国骨伤,1999,12(2)：49-51。
[13]吕红斌,胡建中,罗承耀等。川芎嗪关节腔内注射对膝骨关节炎的治疗作用[J]。中南大学学报(医学版),2006,31(4)：591-594。
[14]刘舟。从《伤寒论》中的“支节颇疼”进而探讨《内经》中的“少阳主骨”[J]。光明中医杂志,2004,19(6)：2。
[15]谢健。足少阳主骨所生病在针灸临床的应用[J]。云南中医杂志,1994(06)：25-30。
[16]刘永臣。由柴胡桂技汤治“支节烦疼”体会“少阳主骨”[J]。中国中医药报,2005,4(4)。
[17]徐叔云。药理学实验方法[M]。北京：人民卫生出版社,2002,203-204。
[18]Manning WK, Bonner WM Jr.Isolation and culture of chondrocytes fromhuman articular cartilage [J]. Arthritis Rheum,1967,10(1):235-239.
[19]李元,杨庆铭,徐建强等。关节软骨细胞分离—胶原酶的细胞毒性作用初探[J]。上海医学,1998；21(2)：81-83。
[20]郝鹏,李胜富,裴福兴等。成年兔关节软骨创伤模型中软骨细胞的分离及体外培养观察[J]。华西医学,2007；22(1)：108-110。
[21]李昊。不同年龄兔软骨细胞培养和形态学研究[J]。山西职工医学院学报,2008：18(1)：15-17。
[22]Kurz B, Domm C, Jin M, et al. Tissue engineering of articular cart ilageunder the influence of collagen I/III membranes and low oxygen tens ion[J]. Tissue Eng,2004;10 (78):1277.
[23]Kart sogiannis V, Ng KW. Cell lines and primary cell cultures in the study of bone cell biology[J]. Mol Ceil Endoerinol,2004; 228(122): 79-82.
[24]夏万尧,曹谊林,商庆新等。组织工程化软骨组织形成的最佳细胞浓度和最佳形成时间的实验研究[J]。中国修复重建外科杂志,1999,13(4)：244-248。
[25]夏青,王卫东,魏振等。兔关节软骨细胞的体外培养及鉴定[J]。中国医师杂志,2007：9(9)：1174-1177。
[26]Dading EM, Athanasiou KA. Rapid phenotypic changes in passaged articular chondrocyte subpoulations[J]. Ortho Res,2005; 23(2):425-432.
[27]Schulze-Tanzil G, de Souza P, Villegas Castrejon H, et al. Rediffrentiation of dedifferentiated human chondrocytes in high-density cultures. Cell Tissue Res,2002; 308(3):371-379.
[28]Goessler UR, Bugert P, Bieback K, et al. Expression of collagen and tiber-associated proteins in human septal cartilage during in vitro dedifferentiation. Int J Mol Med,2004; 14(6):1015-1022.
[29]张群豪,陈可冀。血清药理学在中药及复方研究中应用的评价[J]。中国中西医结合杂志,1996；16(3)：131-133。
[30]杨奎,张德波,史焱等。含黄芩血清及黄芩甙影响内生致热产生的研究[J]。中药药理与临床,1994；13(6)：1410-1413。
[31冯伟。MTT法分析中药含药血清对体外软骨细胞增殖影响的研究[J]。上海中医药大学学报,2000；14(1)：43-45。
[32]崔晓兰,贺玉琢,肖英杰等。中药药理研究的新思路。中国中医药科技,1997；4(4)：239-241。
[33]冯伟,石印玉,沈培芝等。补肾方和补肝方含药血清对软骨细胞增殖动力学研究[J]。浙江中医学院学报,2001,25(4)：15-17。
[34]张荣华,舒晓春。中药复方补肾活血液对成骨细胞影响的实验研究[J]中国病理生理杂志,2003,19(6)769-772。
[35]许勇,杜宁,符诗聪等。健骨颗粒含药血清对人成骨细胞增殖芳香化酶活性的影响[J]。中国骨肿瘤骨病,2003,2(1)：51-54。
[36]刘明怀。黄芪注射液对病毒性心肌炎TNF-A、IL-1和IL-6的影响[J]。实用中医药杂志,2006,22(6)329-330。
[37]刘平,王宁生,雷燕等。关于血清药理学的若干思考[J]。中国中西医结 合杂志,1999：10(5)：263-266。
[38]秦梦,王和鸣,娄玉钤。不同浓度骨痹舒片含药血清对体外培养软骨细胞增殖的影响[J]。中国骨伤,2011,24(10)841-844。
[39]吕红斌,岳珍,王嘉芙。4种中药对体外培养兔关节软骨细胞代谢的影响[J]。中国运动医学杂志杂志,1995；14(3)：135-137。
[40]Kim SJ, Ju Jw, Oh CD, Yoon YM, Song WK, et al. TRK-1/2 and p38 kinase oppositely regulate nitric oxide-induced apoptosis of chondrocytes in association with p53 caspase-3 and differentiation status[J]. J Biol Chem,2002,277(2):1332-1339.
[41]Osaki M, Tan L, Choy BK, et al. The TATA-containing core promoter of the type II collagen gene(COL2A1) is the target of interferon gammamediated inhibition in human chondrocytes requirement for Statlalpha, Jakl and Jak2[J]. Biochem J,2003; 369(81):103-115.
[42]孙明林,朱雷,吕丹。Ⅱ型胶原蛋白对兔去分化软骨细胞再分化的作用[J]。中国修复重建外科杂志,2010,24(10)：1244-1247。
[43]刘瑾春,刘霞,曹谊林。软骨细胞培养中细胞去分化和再分化研究进展。中华整形外科杂志,2011,7,27(4)：318-320。
[44]Grigolo B, Lisignoli G,Piacentini A,etc. Evidence for redifferentiation of human chondrocytes grown on a hyaluronan-based biomaterial(HYAff 11):molecular, immunohistochemical and ultra-structural analysis Biomaterials.2002,23(4):1187-1195.
[45]Mukaida T, Urabe K, Naruse K, etc. Influence of three dimensional culture in a type II collagen sponge on primary cultured and dedifferen-tiated chondrocytes. Journal of Orthopaedic Science.2005,10(5):52.
[46]王卫国,娄思权,佘华等。TGF-β2转染关节软骨细胞的实验研究.中华骨科杂志,2003,7,23(7)：434-438。
[47]Kaps C, Bramlage C, Smolian H, etal. Bone morphogenetic proteins promote cartilage differentiation and protect engineered artificial cartilage from fibroblast invasion and destruction. Arthritis Rheum.2002,46(1):149-162.
[48]Messner K, Maletius W. The long-term prognosis for severe damage to weight-bearing cartilage in the knee:a 14 year clinical and radiographic follow-up in 28 young athletes. Acta Orthop Scand 67,165, 1996.
[49]Gotterbarm T, Breusch SJ, Schneider U, etal. The minipig model for experimental chondral and osteochondral defect repair in tissue engineering:retrospective analysis of 180 defects. Lab Anim 42,71, 2008.
[50]Wei X, Messner K. Maturation dependent durability of spontaneous cartilage repair in rabbit knee joint[J]. Jbiomed Mater Res,2004,46: 539-548.
[51]陈志群。针药并用治疗膝关节骨性关节炎50例临床观察[J]。中医药导报,2010,16(12)：72—73。
[52]Breinan HA, Mins TH, et al. Effect of cultured autologous chondrocytes on repair of chondral defect s in a canine model. J Bone Joint Surg (Am),1997,97:1439-1451.
[53]Gross RE. Repair of cartilage defects in the knee[J]. J Knee Surg, 2002,15(3):167-169.
[54]Buckw alt er JA, Rosenberg L, Cout ts R, etal. Articular cartilage: Injury and repair. Injury and Repair of the Musculoskeletal Soft Tissues 1988,465-482.
[55]胡炯,杜宁,代岭辉等。建立膝骨性关节炎关节软骨缺损的动物模型的研究[J]。中国中医骨伤科杂志,2009,17(11)：13-16。
[56]Mankin HJ, Mow VC, Buckwalter JA, et al. Format ion and function of arthcular cartilage. In:Sheldon R, Simon MD, eds. Orthopaedic BasiC Science. Rosemont, IL, American Academy of Orthopaedic Surgeons,1994, pp447-486.
[57]Allan DA. Structure and physiology of joints and their relationship to repetitive strain injuries. Clinc Orthop 351:32-38,1998.
[58]Tripple SB. Articular cartilage research. Curr Opin Rheumatol 2: 777-782,1990.
[59]Eyre D, Wu J. Collagens structure and cartilage matrixin tegrity. J Rheumatol,1995,22:82-85.
[60]Mandelbaum BR, Browne JE, Fu F, et al. Articular cartilage lesions of the knee。 Am J of Spor Med,1998,26(6):853-861.
[61]Heard F, Heard A,Harmand MF. Apoptosis in normal and osteoarthritic human articular cartilage[J]. Ann Rheum DiS,2000,59(12):959-965.
[62]Huber M, Trattnig S, Linner F. Invest Radiol,2000,35(10):573-580.
[63]Dodds RA, Merru K, littlewood A, et al. Expression of mRNA for IL-lb, IL1-6 and TGF b 1 in developing human bone and cartilage. J Histoehem Cytoehem,1994,42:733-744.
[64]Yamashita F, Sakakida K, Kusuzaki K, et al. Immunohistoehermical localization of interleukin-1 in human growth cartilage. Nippon Seikeigeka Gakkai Zasshi,1989,63:562-568.
[65]Dodds RA, Merry K, Littlewood A, et al. Expression of mRNA for IL-1 β, IL-6 and TGF B1 in developing human bone and cartilage. J Histochem Cytochem,1994,42:733-744.
[66]Teshima S, Nakanishi H, Nishizawa M, et al. Up-regulation of IL-lreceptor through PI3K/Akt is essential for the induction of iNOS gene expression in hepatocytes [J]. Hepatology,2004,40 (4):616-623.
[67]Kolettas E. Muir HI, Barrett JC, et al. Chondrocyte phenotype and cellsurvival are regulated by culture conditions and by specific Cytokines through the expression of sox-9 transcription factor. Rheumatology (oxford),2001,40:1146-1156.
[68]高洁生。骨关节炎治疗新视角[J]。中华风湿病学杂志,2008,12(12)：801-802。
1. Losina E, Walensky RP, Kessler CL, et al. Cost effectiveness of total knee arthroplasty in the United States:patient risk and hospital volume [J].Arch Intern Med.2009 Jun 22;169(12):1113-1121.
2. Ritzker KPH, Gay S, Jimenez SA. Osteoarthritis cartilage histo-pathology:grading and staging[J]. Osteoarthritis Cartilage,2006, 14:13-29.
3.袁淑娟,梁英,薛燕萍等。感觉运动训练治疗膝关节骨性关节炎的疗效观察。中华物理医学与康复杂志,2011,4；33(4)290-293。
4.赵焱全,加味四妙散为主治疗膝关节慢性滑膜炎体会[J]。中国现代药物应用,2009,3(18)：105—106。
5.金军,中药热敷治疗膝关节骨性关节炎180例临床观察[J]。中国中医基础医学杂志,2010,16(4)：344—346。
6.李俐,吴岩,何广富.血府逐瘀汤加减治疗骨性关节炎53例[J]。长春中医药大学学报,2007,23(6)：47。
7.何庆勇,张吉。针刺配合中药内服治疗膝骨性关节炎的临床观察[J]。时珍国医国药,2007,5(1)：36。
8.张智芳、王海明。温针灸治疗膝关节骨性关节炎的疗效观察。国际中医中药杂志,2010,32(3)：243-244。
9.王思茹,郭新惠,刘雪庆,发泡疗法治疗膝关节骨性关节炎的效果观察。国际护理学杂志,2011,29(7)1101-1103。
10.周兰芳,陈勇嫦,莫少媛,中药熏洗配合手法按摩治疗膝骨性关节炎的临床护理观察,国际医药卫生导报,2011,17(19)：2427-2429。
11. Haflah NH, Jaarin K, Abdullah S, et al. Palm vitamin E and glucosamine sulphate in the treatment of osteoarthritis of the knee. Saudi Med J,2009,30(11):1432-1438.
12. Black C, Clar C, Henderson R, et al. The clinical effectiveness of glucosamine and chondroitin supplements in slowing or arresting progression of osteoarthritis of the knee:a systematic review and economic evaluation. Health Technol Assess,2009,13(52):1.148.
13.江相保,高李侠。盐酸氨基葡萄糖治疗膝退行性骨性关节炎的临床观察。中国修复重建外科杂志,2008,22(1)：29-31。
14. Hempfling H. Intr-articulal hyalaronic acid after knee Arthro-scopy:a two-year study. Knee Surg Sports Traumatol Arthrosc,2007, 15:537-546.
15. Diracoglu D, Vural M, Baskent A, et al. The effect of viscosupple-mentation on neuromuscular control of the knee in patients with osteoarthritis. J Back Musculoskelet Rehabil,2009,22:1-9.
16.杨听,曹永平,刘超,文立成等。二膦酸盐对佐剂性关节炎的影响。中国实验外科杂志,2008,25(6)
17. Van Offel JF,Dombrecht EJ, Bridts CH, et al. Influence of bisphosphonates on the production of pro—inflammatory cytokines by activated human articular chondrocytes. Cytokine.2005,31(4): 298-304.
18. Hayami T, Pickarski M, Wesolowski GA, et al. The role of subchondral bone remodeling in osteoarthritis:reduction of cartilage degeneration and prevention of osteophyte formation by alendronate in the rat anterior cruciate ligament transection model. Arthritis Rheum,2004,50(4):1193-1206.
19. Ding M, Danielsen CC, Hvid I. The effects of bone remodeling inhibition by alendronate on three dimensional micro arthitecture of subchondral bone tissues in guinea pig primary osteoarthrosis [J].Calcif Tissue Int.2008,82(1):77-86.
20.王予彬,王惠芳,朱文辉等。膝关节骨性关节炎关节镜下清理和联合髌外侧减压及康复治疗临床疗效的对比研究。中国微创外科杂志,2008,8：678-682。
21.潘惠娟,王予彬,王惠芳,卢亮宇。关节镜下清理髌外侧减压术配合康复训练治疗膝骨性关节炎的疗效观察。中华物理医学与康复杂志,2011,33(2),131-133。
22.余正红,蔡胥等。 膝关节神经分布的解剖学研究及其临床意义[J]。中国临床解剖学杂志,2008,26(1)：11-16。
23.肖春苟。兔膝关节神经支选择性切断后对关节软骨的影响[J]。解剖学研究杂志,2010,4(N)：13-15。
24. Pillings A, Scott DF, Camargo MP, et al. J Bone Joint surg Am, 2000; 82(1):70-79.
25. Zhim F. Laflamme G Y,Viens H, et al. Clin Biomech (Bristol Avon), 2005; 20 (8):871-876.
26. Trieb K, Grohs J, HansLik-Schnabel B, et al. Knee Surg Sports Traumatol Arthrosc,2006; 14(2):149-152.
27. Paccola CA, Fogagnolo E knee surg Sports Traumatol Arthrosc.2005,13(1):19-22.
28. Koshino T, Yoshida T。 Ara Y. et al. Fifteen to twenty-eight years' follow-up results of high tibial valgus osteotomy for osteoarthritic knee, Knee,2004,11(6):439-444.
29. Tang WC, Henderson IJ. High tibia] osteotomy: long term survival analysis and patients perspective. Knee,2005,12(6):410-413.
30. Rodriguez JA, Bhende H, Ranawat CS. Total condylar knee replacement: a 20-year Follow-up study. Clin Orthop Relat Res 2001,388:10-17.
31.Riddle DL, Jiranek WA。 McGlynn FJ. Yearly incidence of unicompartmental knee arthroplasty in the United States. J Arthroplasty,2008,23:408-412.
32. Jamali AA, Scott RD, Rubash HE, et al. Unicompartmental knee arthroplasty:past, present, and future. Am J Orthop,2009,38:17-23.
33.张启栋,郭万首,刘朝晖等.胫骨高位截骨与单髁关节置换治疗膝关节单问室骨关节炎的荟萃分析.中华医学杂志,2009,10,89(39)2768-2772。
34.刘朝晖,郭万首,张启栋等.单髁置换与全膝关节置换治疗膝关节单室骨性关节炎为主的早中期疗效比较,中华医学杂志,2010,10,90(37)：2597-2600。
35. Otten R. Van Roermund PM, Picavet HS. Trends in the number of knee and hip arthroplasties:considerably igore knee and hip prostheses due to ostoarthritis in 2030. Ned Tijdschr Geneeskd, 2010,154:A1534.
36. Wright JG, Santaguida PL, Young N. et al. Patient preferences before and after total knee arthroplasty. J Clin Epidemiol,2010,63 (7):774-782.