沙利度胺对大鼠子宫内膜异位症血管生成的实验研究
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摘要
目的：观察子官内膜异位症(Endometriosis, EMs)模型大鼠异位内膜组织中微血管密度(Microvessel Density, MVD)、血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)和肿瘤坏死因子-α(Tumor Necrosis Factor-α, TNF-α)的表达及沙利度胺(Thalidomide, THD)对以上因子的影响,研究沙利度胺抑制异位内膜血管形成的机制,并探讨其与孕三烯酮联合应用对子宫内膜异位组织血管生成和对异位移植物生长的影响及其作用机理,为临床使用沙利度胺治疗EMs提供实验依据。
方法：建模前随机抽取6只大鼠作为正常对照组,不给予任何处理。术前一天给将要造模的大鼠皮下注射己烯雌酚,剂量为0.1 mg·kg~(-1),使手术时大鼠统一处于动情期。按Vernon等的方法建立SD大鼠EMs动物模型,3周后再次剖腹(二次剖腹)测量移植物体积,病理学证实造模成功。成模大鼠随机分为6组：(1)模型对照组：每天腹腔注射生理盐水2ml；(2)孕三烯酮(YSXT)组0.5mg·kg~(-1)·d~(-1);(3)低剂量沙利度胺(LTHD)组：5mg·kg~(-1)·d~(-1);(4)中剂量沙利度胺(MTHD)组20mg·kg~(-1)·d~(-1)；(5)高剂量沙利度胺(HTHD)组：40mg·kg~(-1)·d~(-1);(6)联合用药组：腹腔注射孕三烯酮0.5mg·kg~(-1)·d~(-1)加沙利度胺20mg·kg~(-1)·d~(-1)。大鼠用药剂量根据人与各种动物之间用药剂量换算公式进行换算,各组按剂量每天腹腔注射给药1次,连用4周后处死大鼠,肉眼观察各组大鼠异位移植物血管生长情况,测量体积大小,并取移植物病灶,分别做HE染色病理切片检查及免疫组化染色,采用医学图像分析系统测定VEGF和TNF-a的表达,并通过Ⅷ因子标记异位子宫内膜血管,测定MVD。
结果：
1.成功的建立了移植性EMs大鼠模型,成模率88.9%(40/45)。生长良好的异位病灶表现为隆起的囊泡结构,内为积液,表面有血管走行。病理学检查可见子宫内膜上皮细胞,腺体和间质。
2.各组大鼠第2次剖腹(建模后3周),各组移植物体积与模型对照组相比无明显差异(P>0.05)。相应处理4周后,第3次剖腹各给药组移植物体积缩小,与第2次剖腹时相比,差异有显著性意义(P<0.01)；与对照组第3次剖腹时相比,差异有统计学意义(P<0.05)。THD对移植物体积的影响呈剂量依赖性改变。联合用药组移植物体积与HTHD组无明显差异(P>0.05),但两组均小于其它各组,差异有统计学意义(P<0.05)。
3.模型对照组异位内膜MVD高于正常对照组(P<0.05)。与模型对照组比较,各给药组异位内膜MVD减少(P<0.05),MVD在YSXT、LTHD、MTHD、联合用药及HTHD组依次减少,LTHD组和YSXT组无明显差异(P＞0.05);THD对MVD的影响呈剂量依赖性改变。
4.模型对照组异位内膜VEGF的表达较正常对照组增强,差异有统计学意义(P<0.05)；各给药组异位内膜VEGF的表达低于模型对照组(P<0.05),其中以联合用药组最为明显,LTHD组和YSXT组无明显差异(P＞0.05);THD对VEGF表达的影响呈剂量依赖性改变。
5.模型对照组异位内膜TNF-α的表达明显高于正常对照组(P<0.05)；而各给药组TNF-α的表达低于模型对照组,差异有统计学意义(P＜0.05)；TNF-α的表达在LTHD、MTHD.HTHD.YSXT和联合用药组依次减弱,其中HTHD.YSXT和联合用药组组间无明显差异(P＞0.05);THD对TNF-α表达的影响呈剂量依赖性改变。结论：
1.改良自体移植法可建立大鼠子宫内膜异位症模型。
2.微血管生成,VEGF和TNF-α在子宫内膜异位症的发病中起重要的作用。
3.沙利度胺通过降低子宫内膜异位症模型大鼠异位内膜中MVD.VEGF和TNF-α的表达,抑制新生血管形成而有效抑制模型大鼠子宫内膜异位症的发展。
4.沙利度胺对子宫内膜异位症大鼠异位移植物的抑制作用呈剂量依赖性。
5.沙利度胺与孕三烯酮联合应用对EMs模型大鼠异位移植物的抑制具有协同作用。
OBJECTIVE:
The aim of this investigation was to observe the microvessel density(MVD), the expression levels of vascular endothelial growth factor(VEGF) and tumor necrosis factor-a(TNF-a) in ectopic endometrium of experimental rats, and the effect of Thalidomide on them. To study the anti-angiogenesis mechanisms of thalidomide in ectopic endometrium, explore the contribution of thalidomide combined with gestrinone to endometriosis angiogenesis and growth of ectopic implants and its mechanism of action, and offer more experimental evidence for clinical use of thalidomide.
METHODS:
Normal control group was established by randomly choose 6 rats before operation. In order to let the rats under unified estrus, the rats were given subcutaneous injection of Diethylstilbestrol 0.1 mg kg~(-1) the day before surgery. Endometriosis models of SD rats were established referring to Vernon's methods. After 3 weeks, the size of each implant was measured by laparotomy, and part of the ectopic endometrial tissue was obtained so as to confirm with pathology that the rat model was made successfully. Then model rats were divided randomly into 6 groups that were treated with normal saline(model control), gestrinone 0.5mg·kg~(-1)·d~(-1),thalidomide 5mg·kg~(-1)·d~(-1)(low-dose thalidomide, LTHD), thalidomide 20mg·kg~(-1)·d~(-1)(middle-dose thalidomide, MTHD), thalidomide 40mg·kg~(-1)·d~(-1)(high-dose thalidomide, HTHD), gestrinone 0.5mg·kg~(-1)·d~(-1) and thalidomide 20mg·kg~(-1)·d~(-1)(drug combination), respectively. All the rats were treated everyday for 28 days continuously. Four weeks later, rats were killed. Ectopic uterine tissues were measured again and evaluated morphologically and histologically. MVD was immunohistochemically determined by immunostaining for factorⅧ. The expression of VEGF and TNF-αwas detected in ectopic endbmetrium by immunohistochemical SP technique.
RESULTS:
1. The model of endometriosis in rats was established successfully, and there was 88.9%(40/45) transplant to develop. The endometrial implants in the rats became cystic structures that were composed of endometrium, endometrial gland and stroma. The macroscopic and histological appearance were consistent with endometriosis.
2. No significant difference was found in the volumes of grafts between model control group and each therapy group on the second operation(P>0.05).Treated for 4 weeks, the volume of each therapy group on the third operation was differently reduced compared with the second operation (P<0.05),and was significantly reduced compared with the model control group on the third operation (P<0.05).The effects of thalidomide on the volumes of implants were dose-dependent. There was no difference between drug combination and HTHD group (P>0.05), and both groups reduced than other groups.
3. MVD in the ectopic lesion was higher in model group compared with that of normal endometrium(P>0.05). Compared with model control group, MVD in the ectopic lesion in each treatment group was decreased obviously (P<0.05).The MVD were reduced in gestrinone、LTHD、MTHD、combination and HTHD group in turn. There was no difference between LTHD and gestrinone (P>0.05). Furthermore, the effect of thalidomide on MVD was dose-dependent.
4. Compared with normal endometrium, the expression of VEGF in the ectopic tissue in model control group was higher which was significantly deceased in each therapy group. In addition, the expression of VEGF showed most decreased in the combination group, while no difference between LTHD and gestrinone group (P>0.05). The inhibition of thalidomide on VEGF was dose-dependent.
5. Samples from the model control group showed significantly higher expression levels of TNF-αcompared to samples from the normal control group(P<0.05),while the expression of TNF-αin each treatment group lower than model control group, and differences have statistical significance(P<0.05). the expression of TNF-a was attenuated in LTHD、MTHD、HTHD、gestrinone and combination group in turn. No difference was found among HTFHD、gestrinone and combination group (P>0.05).It was dose-dependent that THD suppress the expression of TNF-a.
CONCLUSIONS:
1. The rat model of endometriosis which reformed and conducted by autotransplantation of its own endometrium was successfully made.
2. It can be deduced that angiogenesis, VEGF and TNF-a play a considerable role in pathogenesis of endometriosis.
3. Thalidomide can decrease the quantity of micro-vessel and the expression levels of VEGF and TNF-a in heterotopic endometrium of endometriosis rats. It could exert an inhitory effect on the development of endometriosis in rats, possibly through suppression of neovascularization.
4. Thalidomide restrained the ectopic endometriotic implants in EMs rats by means of dose-dependent.
5. Thalidomide in combination with gestrinone had synergistic action on the depressant effect on the ectopic implants in EMs rats.
方法：建模前随机抽取6只大鼠作为正常对照组,不给予任何处理。术前一天给将要造模的大鼠皮下注射己烯雌酚,剂量为0.1 mg·kg~(-1),使手术时大鼠统一处于动情期。按Vernon等的方法建立SD大鼠EMs动物模型,3周后再次剖腹(二次剖腹)测量移植物体积,病理学证实造模成功。成模大鼠随机分为6组：(1)模型对照组：每天腹腔注射生理盐水2ml；(2)孕三烯酮(YSXT)组0.5mg·kg~(-1)·d~(-1);(3)低剂量沙利度胺(LTHD)组：5mg·kg~(-1)·d~(-1);(4)中剂量沙利度胺(MTHD)组20mg·kg~(-1)·d~(-1)；(5)高剂量沙利度胺(HTHD)组：40mg·kg~(-1)·d~(-1);(6)联合用药组：腹腔注射孕三烯酮0.5mg·kg~(-1)·d~(-1)加沙利度胺20mg·kg~(-1)·d~(-1)。大鼠用药剂量根据人与各种动物之间用药剂量换算公式进行换算,各组按剂量每天腹腔注射给药1次,连用4周后处死大鼠,肉眼观察各组大鼠异位移植物血管生长情况,测量体积大小,并取移植物病灶,分别做HE染色病理切片检查及免疫组化染色,采用医学图像分析系统测定VEGF和TNF-a的表达,并通过Ⅷ因子标记异位子宫内膜血管,测定MVD。
结果：
1.成功的建立了移植性EMs大鼠模型,成模率88.9%(40/45)。生长良好的异位病灶表现为隆起的囊泡结构,内为积液,表面有血管走行。病理学检查可见子宫内膜上皮细胞,腺体和间质。
2.各组大鼠第2次剖腹(建模后3周),各组移植物体积与模型对照组相比无明显差异(P>0.05)。相应处理4周后,第3次剖腹各给药组移植物体积缩小,与第2次剖腹时相比,差异有显著性意义(P<0.01)；与对照组第3次剖腹时相比,差异有统计学意义(P<0.05)。THD对移植物体积的影响呈剂量依赖性改变。联合用药组移植物体积与HTHD组无明显差异(P>0.05),但两组均小于其它各组,差异有统计学意义(P<0.05)。
3.模型对照组异位内膜MVD高于正常对照组(P<0.05)。与模型对照组比较,各给药组异位内膜MVD减少(P<0.05),MVD在YSXT、LTHD、MTHD、联合用药及HTHD组依次减少,LTHD组和YSXT组无明显差异(P＞0.05);THD对MVD的影响呈剂量依赖性改变。
4.模型对照组异位内膜VEGF的表达较正常对照组增强,差异有统计学意义(P<0.05)；各给药组异位内膜VEGF的表达低于模型对照组(P<0.05),其中以联合用药组最为明显,LTHD组和YSXT组无明显差异(P＞0.05);THD对VEGF表达的影响呈剂量依赖性改变。
5.模型对照组异位内膜TNF-α的表达明显高于正常对照组(P<0.05)；而各给药组TNF-α的表达低于模型对照组,差异有统计学意义(P＜0.05)；TNF-α的表达在LTHD、MTHD.HTHD.YSXT和联合用药组依次减弱,其中HTHD.YSXT和联合用药组组间无明显差异(P＞0.05);THD对TNF-α表达的影响呈剂量依赖性改变。结论：
1.改良自体移植法可建立大鼠子宫内膜异位症模型。
2.微血管生成,VEGF和TNF-α在子宫内膜异位症的发病中起重要的作用。
3.沙利度胺通过降低子宫内膜异位症模型大鼠异位内膜中MVD.VEGF和TNF-α的表达,抑制新生血管形成而有效抑制模型大鼠子宫内膜异位症的发展。
4.沙利度胺对子宫内膜异位症大鼠异位移植物的抑制作用呈剂量依赖性。
5.沙利度胺与孕三烯酮联合应用对EMs模型大鼠异位移植物的抑制具有协同作用。
OBJECTIVE:
The aim of this investigation was to observe the microvessel density(MVD), the expression levels of vascular endothelial growth factor(VEGF) and tumor necrosis factor-a(TNF-a) in ectopic endometrium of experimental rats, and the effect of Thalidomide on them. To study the anti-angiogenesis mechanisms of thalidomide in ectopic endometrium, explore the contribution of thalidomide combined with gestrinone to endometriosis angiogenesis and growth of ectopic implants and its mechanism of action, and offer more experimental evidence for clinical use of thalidomide.
METHODS:
Normal control group was established by randomly choose 6 rats before operation. In order to let the rats under unified estrus, the rats were given subcutaneous injection of Diethylstilbestrol 0.1 mg kg~(-1) the day before surgery. Endometriosis models of SD rats were established referring to Vernon's methods. After 3 weeks, the size of each implant was measured by laparotomy, and part of the ectopic endometrial tissue was obtained so as to confirm with pathology that the rat model was made successfully. Then model rats were divided randomly into 6 groups that were treated with normal saline(model control), gestrinone 0.5mg·kg~(-1)·d~(-1),thalidomide 5mg·kg~(-1)·d~(-1)(low-dose thalidomide, LTHD), thalidomide 20mg·kg~(-1)·d~(-1)(middle-dose thalidomide, MTHD), thalidomide 40mg·kg~(-1)·d~(-1)(high-dose thalidomide, HTHD), gestrinone 0.5mg·kg~(-1)·d~(-1) and thalidomide 20mg·kg~(-1)·d~(-1)(drug combination), respectively. All the rats were treated everyday for 28 days continuously. Four weeks later, rats were killed. Ectopic uterine tissues were measured again and evaluated morphologically and histologically. MVD was immunohistochemically determined by immunostaining for factorⅧ. The expression of VEGF and TNF-αwas detected in ectopic endbmetrium by immunohistochemical SP technique.
RESULTS:
1. The model of endometriosis in rats was established successfully, and there was 88.9%(40/45) transplant to develop. The endometrial implants in the rats became cystic structures that were composed of endometrium, endometrial gland and stroma. The macroscopic and histological appearance were consistent with endometriosis.
2. No significant difference was found in the volumes of grafts between model control group and each therapy group on the second operation(P>0.05).Treated for 4 weeks, the volume of each therapy group on the third operation was differently reduced compared with the second operation (P<0.05),and was significantly reduced compared with the model control group on the third operation (P<0.05).The effects of thalidomide on the volumes of implants were dose-dependent. There was no difference between drug combination and HTHD group (P>0.05), and both groups reduced than other groups.
3. MVD in the ectopic lesion was higher in model group compared with that of normal endometrium(P>0.05). Compared with model control group, MVD in the ectopic lesion in each treatment group was decreased obviously (P<0.05).The MVD were reduced in gestrinone、LTHD、MTHD、combination and HTHD group in turn. There was no difference between LTHD and gestrinone (P>0.05). Furthermore, the effect of thalidomide on MVD was dose-dependent.
4. Compared with normal endometrium, the expression of VEGF in the ectopic tissue in model control group was higher which was significantly deceased in each therapy group. In addition, the expression of VEGF showed most decreased in the combination group, while no difference between LTHD and gestrinone group (P>0.05). The inhibition of thalidomide on VEGF was dose-dependent.
5. Samples from the model control group showed significantly higher expression levels of TNF-αcompared to samples from the normal control group(P<0.05),while the expression of TNF-αin each treatment group lower than model control group, and differences have statistical significance(P<0.05). the expression of TNF-a was attenuated in LTHD、MTHD、HTHD、gestrinone and combination group in turn. No difference was found among HTFHD、gestrinone and combination group (P>0.05).It was dose-dependent that THD suppress the expression of TNF-a.
CONCLUSIONS:
1. The rat model of endometriosis which reformed and conducted by autotransplantation of its own endometrium was successfully made.
2. It can be deduced that angiogenesis, VEGF and TNF-a play a considerable role in pathogenesis of endometriosis.
3. Thalidomide can decrease the quantity of micro-vessel and the expression levels of VEGF and TNF-a in heterotopic endometrium of endometriosis rats. It could exert an inhitory effect on the development of endometriosis in rats, possibly through suppression of neovascularization.
4. Thalidomide restrained the ectopic endometriotic implants in EMs rats by means of dose-dependent.
5. Thalidomide in combination with gestrinone had synergistic action on the depressant effect on the ectopic implants in EMs rats.
引文
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