放疗相关盆腔纤维化的多光子光学诊断及应用吡非尼酮干预的实验研究
发布时间：2018-08-31 07:30
【摘要】：1直肠癌新辅助放化疗后放疗相关性结肠纤维化的多光子光学诊断研究目的:探讨多光子光学诊断技术在直肠癌辅助放化疗后所致的放疗相关性结肠纤维化的诊断作用。材料与方法:回顾性收集我科12例因放射性盆腔软组织纤维化致肠管严重狭窄或肠梗阻患者的手术石蜡标本(纤维化组)以及另外12例直肠癌新辅助放化疗后随访期间未发生明显纤维化的患者的手术石蜡标本(未纤维化组),石蜡标本取肠管近切端,以反映吻合口近端肠管的情况。另收集10例直肠癌新辅助放化疗后患者(放化疗组)以及10例未行新辅助放化疗患者(未放化疗组)的新鲜近切端手术标本。应用多光子光学诊断技术检测实验样本中的胶原密度、胶原纤维方向性指数,同时应用HE染色及VG染色的技术进行对比研究。结果:多光子光学诊断技术可很好地显示实验样本中的胶原纤维及弹性纤维。放疗可导致粘膜下层及固有肌层内的胶原密度增高或出现胶原纤维破坏并出现大量不成熟的新生胶原纤维、粘膜下层内胶原纤维排列紊乱以及弹性纤维的断裂。石蜡标本中两组的粘膜下层内胶原密度含量均高于肌层内的胶原密度(p0.0001),纤维化组粘膜下层内的胶原密度高于未纤维化组(p=0.0016),纤维化组肌层内的胶原密度也高于未纤维化组(p0.0001)。纤维化组内的胶原纤维方向性指数显著高于未纤维化组(p0.0001)。新鲜标本中两组的粘膜下层内胶原密度含量均高于肌层内的胶原密度(p0.0001),放化疗组粘膜下层内的胶原密度高于未放化疗组(p=0.0002),放化疗组肌层内的胶原密度也高于未放化疗组(p=0.0014)。放化疗组内的胶原纤维方向性指数显著高于未放化疗组(p=0.0068)。结论:多光子光学诊断技术可以检测放疗后结肠肠管内的早期纤维化相关病变。2吡非尼酮干预大鼠盆腔纤维化放疗模型的实验研究目的:探讨吡非尼酮干预大鼠盆腔纤维化放疗模型中受照射肠管及肠管周围肌肉组织纤维化的作用。材料与方法:应用SD雄性大鼠构建盆腔放射性纤维化的大鼠模型,并应用吡非尼酮进行干预研究。大鼠随机分为空白对照组(A组)、吡非尼酮干预组(B组)及放射对照组(C组),在放射后10周每组大鼠取材5只,分别取大鼠直肠及直肠周围肌肉组织进行实验。应用多光子活检技术、HE染色及VG染色分析大鼠样品内胶原密度及胶原纤维方向性,RT-PCR及Western检测TGF-β1、Smad3、CTGF等指标的表达情况。结果:实验成功构建大鼠放射性纤维模型。A组及B组中的粘膜下层胶原密度均显著高于肌层内的胶原密度(p均小于0.0001),而A组及B组间粘膜下层的胶原密度含量无显著性差异(p=0.20),但C组粘膜下层胶原密度含量较B组则显著性下降(p=0.004)。C组中可见肌层内的胶原密度含量显著性升高(p=0.0008)。三组方向性指数单因素方差分析比较具有显著性差异(p=0.04)。三组大鼠直肠周围肌肉内的胶原纤维比例分别为0.024±0.002、0.08±0.003和0.14±0.006,单因素方差分析显示三组之间存在显著性差异(p0.0001);荧光定量PCR结果显示三组的直肠(p=0.04)及直肠周围肌肉(p=0.004)内TGF-β1均存在显著性差异,B组内TGF-β1显著下降。Smad3的表达水平在三组见未见显著性差异。单因素方差分析显示CTGF的表达水平在三组间未见统计学差异,但可见C组的表达水平有高于B组的趋势;Western blot检测结果显示C组直肠组织中的TGF-β1水平显著升高(p=0.036);但三组直肠周围肌肉组织内的TGF-β1水平差异无显著性。Smad3表达在三组的直肠及其周围肌肉均未见显著性差异。CTGF水平在B组中的直肠及其周围肌肉内的表达均有显著性下降。结论:吡非尼酮可减轻大鼠盆腔放射模型中的肠管及直肠周围肌肉的纤维化,其可能是通过抑制组织内TGF-β1及CTGF的蛋白质表达起作用,对放射性相关纤维化的形成具有预防作用。多光子活检技术可有效应用于判断大鼠盆腔放疗模型中直肠肠管内粘膜下层及肌层的纤维化情况。3吡非尼酮干预放疗相关盆腔纤维化动物模型的基因表达谱分析目的:分析吡非尼酮对放疗后盆腔纤维化大鼠模型基因表达谱的影响。材料与方法:应用Affymetrix大鼠Genome 230 2.0 Array芯片对比分析空白对照组(A组)、吡非尼酮干预组(B组)及放射对照组(C组)大鼠的直肠组织内基因表达谱变化情况。结果:B组与A组基因表达谱相比发差异表达基因的功能主要是对脂类的反应、有机环化合物的反应、类固醇类激素的反应。C组与A组相比,显著差异基因可以富集到的GO功能与胞外基质相关的排序靠前,说明C组中胞外基质受影响的程度比较明显。B组与C组之间的差异表达基因GO功能富集到胞外空间、胞外基质、细胞迁移等方面。三组之间相比所富集到的GO功能显著不同。吡非尼酮干预后的通路富集与放射对照组显著的不同。结论:三组大鼠直肠标本的基因芯片表达谱对比分析表明,吡非尼酮具有减轻放射线对直肠组织细胞外基质影响的作用,提示吡非尼酮具有预防放疗相关纤维化的作用。
[Abstract]:1. Multiphoton Optical Diagnosis of Radiotherapy-associated Colon Fibrosis after Neoadjuvant Radiotherapy and Chemotherapy for Rectal Cancer Objective: To investigate the diagnostic value of multiphoton optic diagnostic technique in radiotherapy-associated Colon Fibrosis after Neoadjuvant Radiotherapy and Chemotherapy for Rectal Cancer. Surgical paraffin specimens from patients with severe stricture or intestinal obstruction (fibrosis group) and 12 patients with rectal cancer who did not have obvious fibrosis during the follow-up period after neoadjuvant radiotherapy and chemotherapy (non-fibrosis group) were taken from the proximal intestinal canal to reflect the situation of the proximal intestinal canal anastomosis. Fresh proximal resection specimens were obtained from adjuvant radiotherapy and chemotherapy group and 10 patients without neoadjuvant radiotherapy and chemotherapy group. Collagen density and directional index of collagen fibers in the experimental specimens were measured by multiphoton optical diagnostic technique. The collagen fibers and elastic fibers in the experimental samples can be well displayed by the sub-optical diagnostic technique. Radiotherapy can lead to increased collagen density in the submucosa and lamina propria or collagen fibers destruction and a large number of immature new collagen fibers, disorder of collagen fibers arrangement in the submucosa and breakage of elastic fibers. The collagen density in the submucosa of the fibrosis group was higher than that of the non-fibrosis group (p = 0.0016). The collagen density in the fibrosis group was higher than that in the non-fibrosis group (p 0.0001). The collagen Directional Index in the fibrosis group was significantly higher than that in the non-fibrosis group (p 0.0001). In the fresh samples, the collagen density in the submucosa was higher than that in the myometrium (p0.0001), the collagen density in the submucosa in the radiochemotherapy group was higher than that in the non-radiochemotherapy group (p = 0.0002), and the collagen density in the myometrium in the radiochemotherapy group was also higher than that in the non-radiochemotherapy group (p = 0.0014). The directional index was significantly higher than that of the control group (p = 0.0068). CONCLUSION: Multiphoton optical diagnostic technique can be used to detect the early fibrosis-related lesions in the colon and intestine after radiotherapy. 2 Experimental study of pirfenidone on the pelvic fibrosis radiotherapy model in rats Materials and Methods: SD male rats were used to establish the model of pelvic radiation fibrosis, and the intervention study was carried out with pirfenidone. The rats were randomly divided into blank control group (group A), pirfenidone intervention group (group B) and radiation control group (group C). The rats were taken from each group 10 weeks after radiation. Multiphoton biopsy, HE staining and VG staining were used to analyze the density and orientation of collagen fibers in rats. RT-PCR and Western blotting were used to detect the expression of TGF-beta 1, Smad3 and CTGF. The density of collagen in the submucosa of group B was significantly higher than that in the myometrium (p < 0.0001), but there was no significant difference in the density of collagen between group A and group B (p = 0.20), but the density of collagen in the submucosa of group C was significantly lower than that in group B (p = 0.004). (p = 0.0008). There was a significant difference among the three groups by one-way ANOVA (p = 0.04). The percentage of collagen fibers in the rectal muscles of the three groups was 0.024 [0.002], 0.08 [0.003] and 0.14 [0.006], respectively. There was a significant difference among the three groups by one-way ANOVA (p0.0001). The results of fluorescence quantitative PCR showed that there were significant differences among the three groups. TGF-beta 1 in rectum (p=0.04) and perirectal muscles (p=0.004) were significantly different, and TGF-beta 1 in group B was significantly decreased. Smad3 expression level was not significantly different among the three groups. The results of blot showed that the levels of TGF-beta 1 in rectal tissues of group C were significantly higher than those of group B (p=0.036), but there was no significant difference among the three groups. CONCLUSION: Pirfenidone can alleviate the fibrosis of intestinal canal and perirectal muscles in pelvic radiation model rats, which may play a role by inhibiting the expression of TGF-beta 1 and CTGF proteins in tissues and prevent the formation of radiation-related fibrosis. Gene expression profiling analysis of pirfenidone in radiotherapy-related pelvic fibrosis animal models Objective: To analyze the effect of pirfenidone on gene expression profiles in a rat model of pelvic fibrosis after radiotherapy. Materials and Methods: Genome 2302.0 Array microarray was used in Affymetrix rats. Results: The function of differentially expressed genes in group B was mainly to respond to lipids, organocyclic compounds and steroids compared with group A. The GO function enriched by the differentially expressed genes between group B and group C was enriched in extracellular space, extracellular matrix and cell migration. The GO function enriched by the differentially expressed genes between group B and group C was not significantly different from that enriched by the three groups. CONCLUSION: The comparative analysis of gene chip expression profiles of rectal specimens from three groups of rats shows that pirfenidone can alleviate the effect of radiation on extracellular matrix of rectal tissue, suggesting that pirfenidone can prevent radiation-related fibrosis.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.37;R730.55
本文编号：2214309
[Abstract]:1. Multiphoton Optical Diagnosis of Radiotherapy-associated Colon Fibrosis after Neoadjuvant Radiotherapy and Chemotherapy for Rectal Cancer Objective: To investigate the diagnostic value of multiphoton optic diagnostic technique in radiotherapy-associated Colon Fibrosis after Neoadjuvant Radiotherapy and Chemotherapy for Rectal Cancer. Surgical paraffin specimens from patients with severe stricture or intestinal obstruction (fibrosis group) and 12 patients with rectal cancer who did not have obvious fibrosis during the follow-up period after neoadjuvant radiotherapy and chemotherapy (non-fibrosis group) were taken from the proximal intestinal canal to reflect the situation of the proximal intestinal canal anastomosis. Fresh proximal resection specimens were obtained from adjuvant radiotherapy and chemotherapy group and 10 patients without neoadjuvant radiotherapy and chemotherapy group. Collagen density and directional index of collagen fibers in the experimental specimens were measured by multiphoton optical diagnostic technique. The collagen fibers and elastic fibers in the experimental samples can be well displayed by the sub-optical diagnostic technique. Radiotherapy can lead to increased collagen density in the submucosa and lamina propria or collagen fibers destruction and a large number of immature new collagen fibers, disorder of collagen fibers arrangement in the submucosa and breakage of elastic fibers. The collagen density in the submucosa of the fibrosis group was higher than that of the non-fibrosis group (p = 0.0016). The collagen density in the fibrosis group was higher than that in the non-fibrosis group (p 0.0001). The collagen Directional Index in the fibrosis group was significantly higher than that in the non-fibrosis group (p 0.0001). In the fresh samples, the collagen density in the submucosa was higher than that in the myometrium (p0.0001), the collagen density in the submucosa in the radiochemotherapy group was higher than that in the non-radiochemotherapy group (p = 0.0002), and the collagen density in the myometrium in the radiochemotherapy group was also higher than that in the non-radiochemotherapy group (p = 0.0014). The directional index was significantly higher than that of the control group (p = 0.0068). CONCLUSION: Multiphoton optical diagnostic technique can be used to detect the early fibrosis-related lesions in the colon and intestine after radiotherapy. 2 Experimental study of pirfenidone on the pelvic fibrosis radiotherapy model in rats Materials and Methods: SD male rats were used to establish the model of pelvic radiation fibrosis, and the intervention study was carried out with pirfenidone. The rats were randomly divided into blank control group (group A), pirfenidone intervention group (group B) and radiation control group (group C). The rats were taken from each group 10 weeks after radiation. Multiphoton biopsy, HE staining and VG staining were used to analyze the density and orientation of collagen fibers in rats. RT-PCR and Western blotting were used to detect the expression of TGF-beta 1, Smad3 and CTGF. The density of collagen in the submucosa of group B was significantly higher than that in the myometrium (p < 0.0001), but there was no significant difference in the density of collagen between group A and group B (p = 0.20), but the density of collagen in the submucosa of group C was significantly lower than that in group B (p = 0.004). (p = 0.0008). There was a significant difference among the three groups by one-way ANOVA (p = 0.04). The percentage of collagen fibers in the rectal muscles of the three groups was 0.024 [0.002], 0.08 [0.003] and 0.14 [0.006], respectively. There was a significant difference among the three groups by one-way ANOVA (p0.0001). The results of fluorescence quantitative PCR showed that there were significant differences among the three groups. TGF-beta 1 in rectum (p=0.04) and perirectal muscles (p=0.004) were significantly different, and TGF-beta 1 in group B was significantly decreased. Smad3 expression level was not significantly different among the three groups. The results of blot showed that the levels of TGF-beta 1 in rectal tissues of group C were significantly higher than those of group B (p=0.036), but there was no significant difference among the three groups. CONCLUSION: Pirfenidone can alleviate the fibrosis of intestinal canal and perirectal muscles in pelvic radiation model rats, which may play a role by inhibiting the expression of TGF-beta 1 and CTGF proteins in tissues and prevent the formation of radiation-related fibrosis. Gene expression profiling analysis of pirfenidone in radiotherapy-related pelvic fibrosis animal models Objective: To analyze the effect of pirfenidone on gene expression profiles in a rat model of pelvic fibrosis after radiotherapy. Materials and Methods: Genome 2302.0 Array microarray was used in Affymetrix rats. Results: The function of differentially expressed genes in group B was mainly to respond to lipids, organocyclic compounds and steroids compared with group A. The GO function enriched by the differentially expressed genes between group B and group C was enriched in extracellular space, extracellular matrix and cell migration. The GO function enriched by the differentially expressed genes between group B and group C was not significantly different from that enriched by the three groups. CONCLUSION: The comparative analysis of gene chip expression profiles of rectal specimens from three groups of rats shows that pirfenidone can alleviate the effect of radiation on extracellular matrix of rectal tissue, suggesting that pirfenidone can prevent radiation-related fibrosis.
【学位授予单位】：福建医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.37;R730.55
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