生脉成骨胶囊促进血管生成的机理研究与临床观察
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摘要
目的
研究中药生脉成骨胶囊药物血清促进血管生成的作用机理,以及对人脐静脉内皮细胞株分泌内皮素1、前列环素I_2和一氧化氮等血管活性物质的影响。为生脉成骨胶囊临床用于治疗股骨头缺血性坏死,改善股骨头血液供应提供实验依据;并通过临床观察验证生脉成骨胶囊促进坏死股骨头及其周围血管生成的效果。从现代药理学角度阐明“瘀去则新生脉长”的中医基础理论。
方法
在复习治疗性血管生成的研究进展和临床应用的基础之上,结合该项国家自然科学基金(No.30171143)和广东省自然科学基金(No.010251)资助项目已经取得的阶段性成果,本博士课题分为基础实验和临床观察两部分进行研究。
基础实验:应用噻唑蓝(methyl thiazolyl tetrazolium,MTT)比色法研究生脉成骨胶囊药物血清对体外培养的人脐静脉细胞株(ECV304)增殖的影响,利用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测生脉成骨胶囊药物血清作用1h和2h后ECV304细胞培养上清中血管内皮生长因子(vascular endothelial growth factor,VEGF)浓度,采用ELISA法检测生脉成骨胶囊药物血清作用1h和2h后,ECV304细胞培养上清中内皮素1(endothelin-1,ET-1)和前列环素I_2(prostacyclin I_2,PGI_2)浓度,用硝酸还原酶法检测生脉成骨胶囊药物血清作用1h和2h后,ECV304细胞培养上清中一氧化氮(nitric oxide,NO)浓度,应用透射电镜技术观察生脉成骨胶囊药物血清作用12h后各组ECV304细胞的超微结构。所得数据以SPSS10.0软件进行统计分析。
临床观察:将纳入研究的股骨头缺血性坏死患者分为单纯介入治疗组(对照组)和中药加介入治疗组(中药组),通过数字减影血管成像技术(digital substraction angiography,DSA)观察治疗前及治疗2个月后的坏死股骨头及其周围血管成像,利用JVC ky-F30B 3-CCD彩色图像摄录输入仪进行图像采集及Kontron iBAS 2.0全自动图像分析系统计算出治疗前后的股骨头及其周围血管成像区域灰度值,并取其差值,以t检验进行统计分析。
ObjectiveTo study the angiogenesis mechanism of Shengmai Chenggu Capsule (SMCGC) and its effects on the function of vasoactive-substance-secreting of endothelial cell, so as to provide experimental evidence for its clinical application to treat avascular necrosis of femoral head and improve the blood supply of femoral head. Further more, to precisely measure the grey value of blood vessels in and around the necrotic femoral heads before and after the treatment of in taking SMCGC and radiological interventional treatment with the technique of digital substraction angiography.To elucidate the basic theory "to promote generating blood vessels by stimulating the blood circulating" of Traditional Chinese Medicine from the viewpoint of modern pharmacology.MethodOn the basis of reviewing the experimental progress and clinical application of therapeutic angiogenesis, combined with the previous results from the same project sponsored by National Scientific Fund Committee (No.30171143) and Scientific Fund of Guangdong Province (No.010251) respectively, this doctoral program was divided into two parts: basic experiment and clinical study. In the first part, MTT method was used to study the influence of SMCGC-containing rabbits' serum on the proliferation of ECV304 cell. After ECV304 cell being stimulated by SMCGC-containing rabbits' serum for 1 hour and 2 hours, ELISA method was applied to measure the VEGF concentration in the cultured liquid;With the same method, the concentration of ET-1、 PGI_2 in the cultured
liquid was detected;The concentration of NO in the cultured liquid was detected with enzymic method. The micro-structure of ECV304 cell was observed by transmission electronic microscope technique after being treated for 12 hours. In the clinical trial part, patients involved in the study, who suffer from avascular necrosis of femoral head, were divided into two groups, the patients in group one received interventional radiology treatment only, the patients in the other group got interventional radiology treatment as well as SMCGC-intaking, digital subtractive angiography technique was used to observe the vessels in and around the necrotic femoral head before the treatment and 2 months after the treatment, with image-analyzing software the grey value of angiography before and after the treatment was measured, the differences between the relevant were analyzed statistically with t test.ResultIn the experimental part, after ECV304 cell being treated with SMCGC-containing rabbits' serum for 1 hour, the concentration VEGF of the cultured liquid in low-dose-group increased significantly VEGF(P<0.01), two hours later, the VEGF concentration of the cultured liquid in low-dose-group and medium-dose-group increased significantly (P<0.01). After being stimulated with different doses of SMCGC-containing rabbits' serum for 12 hours and 36 hours, the proliferation of ECV304 cell in all groups was not significantly increased(P<0.05), further more, the proliferation of ECV304 cell in medium-dose-rabbit-serum group and high-dose-rabbit-serum group was inhibited significantly after being treated 36 hours(P<0.01). After ECV304 cell being treated with SMCGC-containing rabbits' serum for 1 hour, the concentration of ET-1 in cultured liquid of high-dose-group significantly decreased(P<0.05) ? the concentration of PGI2 in cultured liquid of low-dose-group, medium-dose-group and high-dose-group was significantly decreased(P<0.05), the concentration of NO in all three groups increased (P>0.05);2 hours later, the concentration of ET-1 in cultured liquid of high-dose-group significantly increased (P<0.05), compared with the control group, the concentration of PGI2 in all three groups was still at low level (P<0.05), but the concentration of PGI2 in the
cultured liquid of low-dose-group increased significantly compared with that of medium-dose-group and high-dose-group (P<0.05), the concentration of NO in the cultured liquid of all three groups increased (P>0.05). Compared with blank group, medium-dose-group and high-dose-group, the number of chondriosomes in low-dose-group and Ligustrazine group increased , and the size of chondriosomes in Ligustrazine group became larger, there were more and larger transparent bubbles in the ECV304 cells of high-dose-group than in those of other groups.The clinical study results were as follows: two months after the interventional radiological therapy combined with SMCGC-intaking, the grey value of blood vessels in and around the femoral head increased significantly (P<0.05), the hip pain was relieved and the Harris scores increased significantlyoConclusionThe results of the basic experiment indicated that low dose SMCGC-containing rabbits' serum could stimulate ECV304 cell to secret VEGF significantly after being stimulated 1 hour, both low dose and medium dose SMCGC-containing rabbits' serum could stimulate ECV304 cell to secret VEGF significantly after being stimulated hours, however the SMCGC-containing rabbits' serum of the high-dose-group had no such effect, which elucidates stimulating the endothelial cell to secret VEGF may be the mechanism of promoting angiogenesis of SMCGC, but the effect did not increase with the dose. SMCGC-containing rabbits' serum of all the three groups inhibited the PGh-secreting function of ECV304 cell could explained the mechanism of its anti-inflammation and pain-killing effect;SMCGC-containing rabbits' serum of all the three groups promote ECV304 cell to secret NO, as to the ET-1-secreting function, high dose SMCGC-containing rabbits' serum had a two-direction effect, inhibiting first and promoting later. SMCGC-containing rabbits' serum had no proliferation-promoting effect for the ECV304 cell cultured in vitro and had no obvious influence on the microstructure of the ECV304 cell, while Ligustrazine might do some damage to the chondriosomes of ECV304 cells. The results of clinical study showed that interventional radiological therapy combined with SMCGC-intaking had a better therapeutic
angiogenesis effect than merely interventional radiological therapy, further more, interventional radiological therapy combined with SMCGC-intaking had a better therapeutic outcoming when it came to symptoms-relieving and function-improving. All in all, the conclusion can be drawn that SMCGC stimulates the angiogenesis by promoting the endothelial cell to secret VEGF, it also had a profound influence on the secreting function of other vascular substances such as ET-1, NO and PGh, these results provided a reliable experimental and clinical background to use SMCGC as one effective method to treat avascular necrosis of femoral head.
研究中药生脉成骨胶囊药物血清促进血管生成的作用机理,以及对人脐静脉内皮细胞株分泌内皮素1、前列环素I_2和一氧化氮等血管活性物质的影响。为生脉成骨胶囊临床用于治疗股骨头缺血性坏死,改善股骨头血液供应提供实验依据;并通过临床观察验证生脉成骨胶囊促进坏死股骨头及其周围血管生成的效果。从现代药理学角度阐明“瘀去则新生脉长”的中医基础理论。
方法
在复习治疗性血管生成的研究进展和临床应用的基础之上,结合该项国家自然科学基金(No.30171143)和广东省自然科学基金(No.010251)资助项目已经取得的阶段性成果,本博士课题分为基础实验和临床观察两部分进行研究。
基础实验:应用噻唑蓝(methyl thiazolyl tetrazolium,MTT)比色法研究生脉成骨胶囊药物血清对体外培养的人脐静脉细胞株(ECV304)增殖的影响,利用酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)检测生脉成骨胶囊药物血清作用1h和2h后ECV304细胞培养上清中血管内皮生长因子(vascular endothelial growth factor,VEGF)浓度,采用ELISA法检测生脉成骨胶囊药物血清作用1h和2h后,ECV304细胞培养上清中内皮素1(endothelin-1,ET-1)和前列环素I_2(prostacyclin I_2,PGI_2)浓度,用硝酸还原酶法检测生脉成骨胶囊药物血清作用1h和2h后,ECV304细胞培养上清中一氧化氮(nitric oxide,NO)浓度,应用透射电镜技术观察生脉成骨胶囊药物血清作用12h后各组ECV304细胞的超微结构。所得数据以SPSS10.0软件进行统计分析。
临床观察:将纳入研究的股骨头缺血性坏死患者分为单纯介入治疗组(对照组)和中药加介入治疗组(中药组),通过数字减影血管成像技术(digital substraction angiography,DSA)观察治疗前及治疗2个月后的坏死股骨头及其周围血管成像,利用JVC ky-F30B 3-CCD彩色图像摄录输入仪进行图像采集及Kontron iBAS 2.0全自动图像分析系统计算出治疗前后的股骨头及其周围血管成像区域灰度值,并取其差值,以t检验进行统计分析。
ObjectiveTo study the angiogenesis mechanism of Shengmai Chenggu Capsule (SMCGC) and its effects on the function of vasoactive-substance-secreting of endothelial cell, so as to provide experimental evidence for its clinical application to treat avascular necrosis of femoral head and improve the blood supply of femoral head. Further more, to precisely measure the grey value of blood vessels in and around the necrotic femoral heads before and after the treatment of in taking SMCGC and radiological interventional treatment with the technique of digital substraction angiography.To elucidate the basic theory "to promote generating blood vessels by stimulating the blood circulating" of Traditional Chinese Medicine from the viewpoint of modern pharmacology.MethodOn the basis of reviewing the experimental progress and clinical application of therapeutic angiogenesis, combined with the previous results from the same project sponsored by National Scientific Fund Committee (No.30171143) and Scientific Fund of Guangdong Province (No.010251) respectively, this doctoral program was divided into two parts: basic experiment and clinical study. In the first part, MTT method was used to study the influence of SMCGC-containing rabbits' serum on the proliferation of ECV304 cell. After ECV304 cell being stimulated by SMCGC-containing rabbits' serum for 1 hour and 2 hours, ELISA method was applied to measure the VEGF concentration in the cultured liquid;With the same method, the concentration of ET-1、 PGI_2 in the cultured
liquid was detected;The concentration of NO in the cultured liquid was detected with enzymic method. The micro-structure of ECV304 cell was observed by transmission electronic microscope technique after being treated for 12 hours. In the clinical trial part, patients involved in the study, who suffer from avascular necrosis of femoral head, were divided into two groups, the patients in group one received interventional radiology treatment only, the patients in the other group got interventional radiology treatment as well as SMCGC-intaking, digital subtractive angiography technique was used to observe the vessels in and around the necrotic femoral head before the treatment and 2 months after the treatment, with image-analyzing software the grey value of angiography before and after the treatment was measured, the differences between the relevant were analyzed statistically with t test.ResultIn the experimental part, after ECV304 cell being treated with SMCGC-containing rabbits' serum for 1 hour, the concentration VEGF of the cultured liquid in low-dose-group increased significantly VEGF(P<0.01), two hours later, the VEGF concentration of the cultured liquid in low-dose-group and medium-dose-group increased significantly (P<0.01). After being stimulated with different doses of SMCGC-containing rabbits' serum for 12 hours and 36 hours, the proliferation of ECV304 cell in all groups was not significantly increased(P<0.05), further more, the proliferation of ECV304 cell in medium-dose-rabbit-serum group and high-dose-rabbit-serum group was inhibited significantly after being treated 36 hours(P<0.01). After ECV304 cell being treated with SMCGC-containing rabbits' serum for 1 hour, the concentration of ET-1 in cultured liquid of high-dose-group significantly decreased(P<0.05) ? the concentration of PGI2 in cultured liquid of low-dose-group, medium-dose-group and high-dose-group was significantly decreased(P<0.05), the concentration of NO in all three groups increased (P>0.05);2 hours later, the concentration of ET-1 in cultured liquid of high-dose-group significantly increased (P<0.05), compared with the control group, the concentration of PGI2 in all three groups was still at low level (P<0.05), but the concentration of PGI2 in the
cultured liquid of low-dose-group increased significantly compared with that of medium-dose-group and high-dose-group (P<0.05), the concentration of NO in the cultured liquid of all three groups increased (P>0.05). Compared with blank group, medium-dose-group and high-dose-group, the number of chondriosomes in low-dose-group and Ligustrazine group increased , and the size of chondriosomes in Ligustrazine group became larger, there were more and larger transparent bubbles in the ECV304 cells of high-dose-group than in those of other groups.The clinical study results were as follows: two months after the interventional radiological therapy combined with SMCGC-intaking, the grey value of blood vessels in and around the femoral head increased significantly (P<0.05), the hip pain was relieved and the Harris scores increased significantlyoConclusionThe results of the basic experiment indicated that low dose SMCGC-containing rabbits' serum could stimulate ECV304 cell to secret VEGF significantly after being stimulated 1 hour, both low dose and medium dose SMCGC-containing rabbits' serum could stimulate ECV304 cell to secret VEGF significantly after being stimulated hours, however the SMCGC-containing rabbits' serum of the high-dose-group had no such effect, which elucidates stimulating the endothelial cell to secret VEGF may be the mechanism of promoting angiogenesis of SMCGC, but the effect did not increase with the dose. SMCGC-containing rabbits' serum of all the three groups inhibited the PGh-secreting function of ECV304 cell could explained the mechanism of its anti-inflammation and pain-killing effect;SMCGC-containing rabbits' serum of all the three groups promote ECV304 cell to secret NO, as to the ET-1-secreting function, high dose SMCGC-containing rabbits' serum had a two-direction effect, inhibiting first and promoting later. SMCGC-containing rabbits' serum had no proliferation-promoting effect for the ECV304 cell cultured in vitro and had no obvious influence on the microstructure of the ECV304 cell, while Ligustrazine might do some damage to the chondriosomes of ECV304 cells. The results of clinical study showed that interventional radiological therapy combined with SMCGC-intaking had a better therapeutic
angiogenesis effect than merely interventional radiological therapy, further more, interventional radiological therapy combined with SMCGC-intaking had a better therapeutic outcoming when it came to symptoms-relieving and function-improving. All in all, the conclusion can be drawn that SMCGC stimulates the angiogenesis by promoting the endothelial cell to secret VEGF, it also had a profound influence on the secreting function of other vascular substances such as ET-1, NO and PGh, these results provided a reliable experimental and clinical background to use SMCGC as one effective method to treat avascular necrosis of femoral head.
引文
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