三维适形调强放疗对新疆骨包虫的杀灭作用及其机制

The existing medications, surgeries and other treatments to echinococcosis of bone are of poor curative effect, high recurrence rate and disability rates. Although the healing effect and the possible mechanism of IMRT (Intensity Modulated Radiation Therapy) for echinococcosis of bone are still unknown, IMRT could serve ideally as a radical and non-invasive radiotherapy for this disease. The results in the pre-experiment indicate that IMRT has a good treating effect on echinococcosis of bone, but it still needs a further research. Therefore, the author in this research puts forward the hypothesis that the killing effect of IMRT for bone hydatid should be achieved by promoting hydatid germinal cells apoptosis directly, inhibiting the activity of protoscoleces enzyme and reactivating the immune function of the parasitifer. In order to verify the hypothesis, the research will adopt the models of germinal cell of bone hydatid, protoscolex and rats with bone hydatid by means of IMRT, and the killing effects of IMRT on echinococcosis of bone will be discussed from molecules, cells, tissues, and overall aspects of animals with the help of the treatments of MTT、Western Blot、RT-PCR、ELISA、Meisel and Gomori, meanwhile, the Immune-Killing mechanism of "Ray- Immunological Response of the parasitifer- Killing Hydatid" will be discussed also to provide the scientific evidence for the radiotherapy of the echinococcosis of bone.

针对骨包虫病，现有的药物、手术等治疗手段疗效差、复发率及致残率高。采用三维适形调强放疗（IMRT）对骨包虫实施根治性、无侵入性放疗，是一个较为理想的治疗方法，但迄今IMRT对骨包虫的杀灭效果、可能的机制人们仍知之甚少，目前尚无成熟的报道。 我们预实验的结果提示IMRT对骨包虫有很好的杀灭效果，但其作用机制还有待进一步探讨，为此我们提出假说:IMRT对包虫的杀灭作用是通过直接促进包虫生发细胞凋亡、抑制原头节酶的活性、以及重新激发宿主的免疫功能实现的。为验证这一假说，我们将以骨包虫生发细胞、原头节、骨包虫大鼠为模型，对其实施IMRT，采用MTT、Western Blot、RT-PCR、ELISA、Meisel、Gomori等手段从分子、细胞、组织以及动物整体水平多方面探讨IMRT对骨包虫的杀灭效果，并探讨其"射线―宿主免疫应答―杀灭包虫"的免疫杀灭机制，为临床骨包虫病的放射治疗提供科学依据。

本课题通过1、分离、体外培养骨包虫生发细胞，经40Gy的IMRT处理细胞后，MTT法观察IMRT对骨包虫生发细胞增殖的影响；电镜、流式细胞仪观测IMRT对骨包虫细胞凋亡的影响；Western Blotting检测IMRT作用后细胞内Caspase-3蛋白酶活性；RT-PCR检测凋亡相关基因Caspase-3表达。2、分离、体外培养骨包虫原头节，台盼蓝排斥法确定骨包虫原头节死亡率，观察40Gy的IMRT对骨包虫原头节后的即刻杀灭作用；并通过返种实验，了解IMRT对骨包虫原头节感染能力的影响；同时行酶化学染色检测原头节酸性磷酸酶、葡萄糖6-磷酸酶和琥珀酸脱氢酶的活性变化。3、用骨包虫的原头节接种子午沙鼠腿骨，成功建立子午沙鼠继发性骨包虫病动物模型；将80只子午沙鼠分为空白对照组及IMRT组，空白对照组接受0Gy的IMRT假照射，IMRT组40Gy的IMRT照射，照射结束后，子午沙鼠继续饲养1月，通过比较空白对照组和IMRT组的子午沙鼠骨包虫包囊减重率、囊壁的形态改变、TUNEL法检测囊壁细胞凋亡评价IMRT杀伤骨包虫的效果，ELISA、RT-PCR检测子午沙鼠继发性骨包虫病动物模型免疫功能的改变。通过检测IMRT对于骨包虫生发层细胞、局部结构原头节、整个体内骨包虫三个层面的杀灭作用，及IMRT作用后子午沙鼠免疫功能的改变，从分子、细胞、组织以及动物整体水平：1.明确了剂量在40Gy以上的 IMRT在体外对生发细胞、原头节这两种骨包虫组成成分有极强的直接急性杀伤作用和诱导凋亡的作用；2.检测并确定了剂量在40Gy以上的 IMRT对骨包虫在子午沙鼠动物模型体内有极高的杀灭率，能够骨包虫在动物模型体内的生长；3.证明了IMRT对骨包虫的治疗的杀灭机制不但与其高能效应直接破坏骨包虫相关，而且存在：“射线-激发宿主免疫应答-杀灭包虫”的免疫杀灭机制。为临床骨包虫病的放射治疗提供科学的依据。