透骨消痛胶囊对退变软骨细胞增殖影响的实验研究
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摘要
目的：观察透骨消痛胶囊含药血清对正常及退变软骨细胞增殖速率和细胞周期分布的影响,从细胞学的角度上探讨透骨消痛胶囊对正常及退变软骨细胞增殖的干预作用。
方法：1.含药血清及不含药血清的制备：取健康六月龄新西兰大白兔16只,雌雄各半,随机分成2组,即模型组与给药组。给药组予以透骨消痛胶囊水溶液灌胃,模型组则以等量的生理盐水灌胃。取模型组与给药组动物血清制备含透骨消痛胶囊含药血清和不含药血清。
2.含药血清和不含药血清培养第3代软骨细胞：取健康六月龄新西兰大白兔16只,雌雄各半,随机分成2组,即正常组与造模组,通过改良Hulth法,对造模组动物实施造模手术。喂养1周后,开始强迫造模组兔每日活动1次,每次1h。8周后处死动物,分离并培养正常组和造模组动物软骨细胞,培养至第三代,用甲苯胺蓝染液对其进行鉴定。取第3代正常及退变软骨细胞用含药血清与不含药血清进行培养,分为A组(正常软骨细胞不含药血清组)、B组(正常软骨细胞含药血清组)、C组(退变软骨细胞不含药血清组)、D组(退变软骨细胞含药血清组)。用光镜对增殖的软骨细胞进行组织形态学观察,MTT法测细胞相对数,用流式细胞仪测软骨细胞周期分布。结果：1.倒置显微镜观察血清培养后的正常及退变软骨细胞形态,增殖后的正常软骨细胞呈多角形或长梭形,细胞核清晰；增殖后的退变软骨细胞呈多角形或长梭形,部分细胞核固缩,有的偏向一侧。
2.MTT检测显示,各组软骨细胞增殖速度B组>A组>D组>C组,其中B组>A组(P<0.05),B组>C组(P<0.05),B组>D组(P<0.05),A组>C组(P<0.05),A组>D组(P<0.05),D组>C组(P<0.05)。
3.流式细胞仪检测显示,各组软骨细胞周期分布随着时间的变化以及血清干预而发生变化。干预24、48、72h,B组含药血清S、G2/M期比例上升及PI升高高于同期A组不含药血清(P<0.05),D组含药血清S、G2/M期比例上升及PI升高高于同期C组(P<0.05),C、D组退变软骨细胞的S、G2/M期比例、PI值均低于A、B组正常软骨细胞(P<0.05)。
结论：透骨消痛胶囊含药血清能促进正常及退变软骨细胞的增殖,但不能完全使退变软骨细胞达到正常软骨细胞的增殖水平。
Objective:Observe the effects on the proliferation of the degenerate chondrocytes with Tougu XiaoTong Capsule-containing serum, from the perspective of cytology to study the effects of Tougu XiaoTong Capsule interfere the normal and degenerate chondrocytes.
Methods:1. Serum containing drug and serum not containing drug preparation: Sixteen healthy rabbits of New Zealand aged six months, half male and half female were randomly divided into two groups, i.e:the model group and the simva group.The animals of the simva group were enforce intragastric administration with the water-solution of Tougu XiaoTong Capsule, and The animals of the model group were enforce intragastric administration with the isotonic Na chloride.Four days later, we need collect serum containing Tougu XiaoTong Capsule and serum not containing drug.
2. Sixteen healthy rabbits of New Zealand aged six months, half male and half female were randomly divided into two groups, i.e:the normal group and the model group.According to the improved operational method of Hulth, we had operated the animals of the model group.The rabbits of the model group were forced to locomote an hour afer the were fed adaptatively a week ago.Afert eight weeks, rabbits were sacrificed and isolated the chondrocytes from the rabbits knee joint cartilage to culture. Cultured chondrocytes to the 3rd Generation and identified them with toluidine blue. Cultured the 3rd generation normal and degenerate chondrocytes with the serum containing Tougu XiaoTong Capsule and serum not containing drug, divided into four groups, i.e:A (serum containing drug for normal chondrocytes)、B (serum not containing drug for normal chondrocytes)、C(serum containing drug for degenerate chondrocytes)、D (serum not containing drug for degenerate chondrocytes) group.We get to observe the histomorphology of the chondrocytes with the light microscope, detect the chondrocytes'proliferation with MTT and the cycle distribution of the chondrocytes with flow cytometer.
Results:1. Observe the morphology of normal and degenerate chondrocytes with seroculture under the inverted microscope.The proliferating normal chondrocytes display long polygon or spindle and nucleus clearly. The proliferating degenerate chondrocytes display long polygon or spindle, some nuclear condensation and some nuclear defect.
2.MTT test showed that the chondrocytes'proliferation rate in each group were B group> A group> D group> C group, which B group> A group (P<0.05), B group> C group (P<0.05), B group> D group (P<0.05), A group> C group (P<0.05), A group> D group (P<0.05) and D group> C group (P<0.05).
3. Flow cytometer test showed that the cycle distribution of the chondrocytes in each group changed with the change of time and serum intervention.The proportion of S、G2/M and PI in The B group which with the serum containing Tougu XiaoTong Capsule were higer than the A group(P<0.05). The proportion of S、G2/M and PI in The D group which with the serum containing Tougu XiaoTong Capsule were higer than the C group(P<0.05). The proportion of S、G2/M and PI of the degenerate chondrocytes in C、D groups was lower than the normal chondrocytes in A、B groups (P<0.05).
Conclusion:Tougu XiaoTong Capsule can promote the proliferation of the normal and degenerate chondrocytes, But it can not completely make up the degenerate chondrocytes come up to the proliferation rate of normal chondrocytes.
方法：1.含药血清及不含药血清的制备：取健康六月龄新西兰大白兔16只,雌雄各半,随机分成2组,即模型组与给药组。给药组予以透骨消痛胶囊水溶液灌胃,模型组则以等量的生理盐水灌胃。取模型组与给药组动物血清制备含透骨消痛胶囊含药血清和不含药血清。
2.含药血清和不含药血清培养第3代软骨细胞：取健康六月龄新西兰大白兔16只,雌雄各半,随机分成2组,即正常组与造模组,通过改良Hulth法,对造模组动物实施造模手术。喂养1周后,开始强迫造模组兔每日活动1次,每次1h。8周后处死动物,分离并培养正常组和造模组动物软骨细胞,培养至第三代,用甲苯胺蓝染液对其进行鉴定。取第3代正常及退变软骨细胞用含药血清与不含药血清进行培养,分为A组(正常软骨细胞不含药血清组)、B组(正常软骨细胞含药血清组)、C组(退变软骨细胞不含药血清组)、D组(退变软骨细胞含药血清组)。用光镜对增殖的软骨细胞进行组织形态学观察,MTT法测细胞相对数,用流式细胞仪测软骨细胞周期分布。结果：1.倒置显微镜观察血清培养后的正常及退变软骨细胞形态,增殖后的正常软骨细胞呈多角形或长梭形,细胞核清晰；增殖后的退变软骨细胞呈多角形或长梭形,部分细胞核固缩,有的偏向一侧。
2.MTT检测显示,各组软骨细胞增殖速度B组>A组>D组>C组,其中B组>A组(P<0.05),B组>C组(P<0.05),B组>D组(P<0.05),A组>C组(P<0.05),A组>D组(P<0.05),D组>C组(P<0.05)。
3.流式细胞仪检测显示,各组软骨细胞周期分布随着时间的变化以及血清干预而发生变化。干预24、48、72h,B组含药血清S、G2/M期比例上升及PI升高高于同期A组不含药血清(P<0.05),D组含药血清S、G2/M期比例上升及PI升高高于同期C组(P<0.05),C、D组退变软骨细胞的S、G2/M期比例、PI值均低于A、B组正常软骨细胞(P<0.05)。
结论：透骨消痛胶囊含药血清能促进正常及退变软骨细胞的增殖,但不能完全使退变软骨细胞达到正常软骨细胞的增殖水平。
Objective:Observe the effects on the proliferation of the degenerate chondrocytes with Tougu XiaoTong Capsule-containing serum, from the perspective of cytology to study the effects of Tougu XiaoTong Capsule interfere the normal and degenerate chondrocytes.
Methods:1. Serum containing drug and serum not containing drug preparation: Sixteen healthy rabbits of New Zealand aged six months, half male and half female were randomly divided into two groups, i.e:the model group and the simva group.The animals of the simva group were enforce intragastric administration with the water-solution of Tougu XiaoTong Capsule, and The animals of the model group were enforce intragastric administration with the isotonic Na chloride.Four days later, we need collect serum containing Tougu XiaoTong Capsule and serum not containing drug.
2. Sixteen healthy rabbits of New Zealand aged six months, half male and half female were randomly divided into two groups, i.e:the normal group and the model group.According to the improved operational method of Hulth, we had operated the animals of the model group.The rabbits of the model group were forced to locomote an hour afer the were fed adaptatively a week ago.Afert eight weeks, rabbits were sacrificed and isolated the chondrocytes from the rabbits knee joint cartilage to culture. Cultured chondrocytes to the 3rd Generation and identified them with toluidine blue. Cultured the 3rd generation normal and degenerate chondrocytes with the serum containing Tougu XiaoTong Capsule and serum not containing drug, divided into four groups, i.e:A (serum containing drug for normal chondrocytes)、B (serum not containing drug for normal chondrocytes)、C(serum containing drug for degenerate chondrocytes)、D (serum not containing drug for degenerate chondrocytes) group.We get to observe the histomorphology of the chondrocytes with the light microscope, detect the chondrocytes'proliferation with MTT and the cycle distribution of the chondrocytes with flow cytometer.
Results:1. Observe the morphology of normal and degenerate chondrocytes with seroculture under the inverted microscope.The proliferating normal chondrocytes display long polygon or spindle and nucleus clearly. The proliferating degenerate chondrocytes display long polygon or spindle, some nuclear condensation and some nuclear defect.
2.MTT test showed that the chondrocytes'proliferation rate in each group were B group> A group> D group> C group, which B group> A group (P<0.05), B group> C group (P<0.05), B group> D group (P<0.05), A group> C group (P<0.05), A group> D group (P<0.05) and D group> C group (P<0.05).
3. Flow cytometer test showed that the cycle distribution of the chondrocytes in each group changed with the change of time and serum intervention.The proportion of S、G2/M and PI in The B group which with the serum containing Tougu XiaoTong Capsule were higer than the A group(P<0.05). The proportion of S、G2/M and PI in The D group which with the serum containing Tougu XiaoTong Capsule were higer than the C group(P<0.05). The proportion of S、G2/M and PI of the degenerate chondrocytes in C、D groups was lower than the normal chondrocytes in A、B groups (P<0.05).
Conclusion:Tougu XiaoTong Capsule can promote the proliferation of the normal and degenerate chondrocytes, But it can not completely make up the degenerate chondrocytes come up to the proliferation rate of normal chondrocytes.
引文
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