摘要
目的：
本课题拟从细胞调亡、免疫细胞因子角度对损伤血瘀证进行研究,为今后临床上从骨骼肌细胞调亡角度、免疫角度有效诊断损伤血瘀证不同病理周期及合理把握用药时期提供实验室依据。通过本论文的探索及研究,不仅能为损伤血瘀证的证候客观化作出重要的贡献,更加能补充说明中医证候学的科学内涵,即宏观与微观的统一,局部与整体的统一,静态与动态的统一。
方法：
SD大鼠80只,按随机原则分为8组,即空白对照组、造模后2h组、造模后8h组、造模后24h组、造模后2d组、造模后3d组、造模后5d组和造模后7d组,每组10只。造模方法采用成熟的定量重力打击法,空白对照组在造模前,其余组分别在相应的时象点处死大鼠,并分离其骨骼肌细胞和血液,测试骨骼肌细胞凋亡情况、病理切片及血液的免疫细胞因子指标IL-2、IL-4、IL-6、IL-8、IL-10和TNF-α,动态观察损伤后骨骼肌细胞凋亡、组织病理状态、全身免疫变化情况,通过损伤血瘀证早期的各种客观化的实验数据,判断损伤血瘀证早期的证本质。同时结合中医诊断理论,通过对中西医的系统比较,探索中医诊断学宏观与微观,局部与整体,静态与动态相统一的理论基础。
结果：
①定量打击法造成的损伤血瘀证必然导致细胞凋亡加速,而凋亡随着时间推移呈现规律性的变化趋势,且这种规律是非直线性的,细胞凋亡水平具有多个进展和稳定周期。
②通过检测阳性率较高的凋亡调节蛋白酶caspase-3,证实凋亡调节蛋白酶caspase-3之间存在多个进展和稳定周期,并与凋亡有着必然的联系。
③通过观察细胞因子IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α,发现损伤血瘀证凋亡变化趋势与机体免疫细胞因子之间存在着双向动态平衡的偶联关系。免疫细胞因子的整体启动点、整体激增点、整体衰落点,只要是发生细胞因子系统性比例失衡,细胞免疫应激状态就迅速加强,进而刺激细胞凋亡的发生。本课题发现了免疫细胞因子系统性消耗造成的凋亡现象,这是科学界的新发现,也从一个侧面反应了中医的阴阳学说理论,并以各种细胞因子与TNF-α之间的比值为例出发,证实与TNF-α相关指标比例的太过与不及皆有可能导致凋亡的加速发生,TNF-α绝不是一个简单的促进细胞凋亡的免疫因子指标。
④通过镜下HE染色,进一步证实出血、免疫反应、凋亡、修复之间具有确切的联动关系,这种关系可以用病理周期明确的描述,并可以解释细胞凋亡4次激增的原因。
⑤从临床意义分析,一周左右的水肿是免疫系统性反应降低导致的细胞凋亡产生的,应该使用扶正药物；闭合性损伤早期理想的治疗方案是减少免疫反应波动。
结论：
损伤血瘀证大鼠存在坏死状凋亡现象而非坏死,其内在的机制与一系列的免疫反应相关。
Purpose:
The topic to be from apoptosis, immune cells the perspective of injury research stasis for the future from a clinical point of skeletal muscle apoptosis, immune injury blood stasis point of effective diagnosis and a reasonable grasp of different pathological cycle of drug use and provide experimental period room basis. Through exploration and research of this thesis, not only for the injury blood stasis syndrome make an important contribution to the objective, and more able to TCM Syndrome supplement the scientific content of the unity of the macro and micro, local and global unity, static and dynamic unity.
Method:
80 SD rats were randomly assigned into 8 groups, namely control group, 2h after the modeling group,8h after the modeling group,24h after the modeling group,2d,3d,5d,7d group after the model, n=10. Using sophisticated modeling of quantitative methods to combat gravity, the control group before modeling, and the remaining groups were as in the corresponding time points rats were killed, and isolated from skeletal muscle cells and blood tests apoptosis of skeletal muscle cells, pathological biopsy and blood indicators of immune cytokines IL-2, IL-4, IL-6, IL-8, IL-10 and TNF-α, dynamic observation of apoptosis in skeletal muscle after injury, pathological states, changes in systemic immune through injury early stasis various objective of the experimental data to determine evidence of injury the nature of early blood stasis. Theory combined with traditional Chinese medicine diagnosis, through the systematic comparison of Chinese and Western medicine, Chinese medicine diagnostics to explore the macro and micro, local and global, static and dynamic unity of the theoretical basis.
Results:
①quantitative methods to combat the damage caused by blood stasis inevitably lead to accelerated apoptosis, and apoptosis was regular and over time the trend, and this law of non-linear in nature, with multiple levels of apoptosis progress and stability cycle.
②higher positive rate by regulation of apoptosis protease caspase-3, confirmed that caspase-3 protease regulation of apoptosis between progress and stability in multiple cycles, and intrinsically linked with apoptosis.
③by observing cytokine IL-2, IL-4, IL-6, IL-8, IL-10, TNF-α, found that apoptosis of blood stasis injury trends and immune cytokines dynamic equilibrium exists between the two-way coupling. The overall factor of immune cells start point, the overall surge in point, the overall decline of points, as long as the occurrence of systemic imbalance of cytokines, immune to the rapid reinforcement of stress, thereby stimulating cell apoptosis. The subject of immune cells found in consumption caused by systemic factors of apoptosis, which is a new scientific discovery, but also from a side reaction of the Chinese theory of yin and yang theory, and various cytokines and the ratio between TNF-αexample starting with TNF-αconfirmed the proportion of relevant indicators is possible too with less acceleration occurs leading to apoptosis, TNF-αis by no means a simple immune factors to promote target cell apoptosis.
④HE staining by microscopy and further confirmed by blood, immune response, apoptosis, repair the linkage between the exact relationship, this relationship can be described clearly pathological cycle and apoptosis may explain the reason for the increase 4 times.
⑤from the clinical significance of analysis, about a week, systemic immune response to reduce edema caused by cells generated righting drugs should be used; closed injury ideal treatment of early immune response is to reduce volatility.
Conclusion: Injury there is blood stasis rats necrosis-like apoptosis, the underlying mechanism is a series of immune response.
引文
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