生肌玉红胶原促血管新生的实验研究
摘要
第一部分研究背景
中医外科古代又名疡科,下肢慢性溃疡是中医外科治疗的优势病种之一,中医在此病的治疗中积累了丰富的临床经验。对于Ⅲ-Ⅳ期慢性下肢溃疡临床多运用生肌玉红膏为代表的药物外用,使得疮面肉芽新鲜,疮面减小。既往我们对生肌玉红膏治疗慢性溃疡进行了临床研究,证明生肌玉红膏可以促进慢性下肢溃疡创面微循环、加速创面肉芽生长及上皮生长,从而促进慢性下肢溃疡创面愈合。
许多研究都在通过调整生肌玉红膏的药物组成来提高疗效。因此,如何通过进一步改良而提高疗效,是中医外科研究的热点之一。本研究拟用生肌玉红膏原方去除轻粉,以减轻其毒性,检测其对血管新生与发生的影响。
高水平、快速的血管化一直是慢性创面修复研究的一个基本课题。应用胶原作为支架载入生长因子制造促血管生长的生物材料植入机体促血管生长为目前国际研究开发的前沿与热点之一。将修饰后的胶原作为减味生肌玉红膏敷料载体,发挥两者促创面愈合优势,是否可以进一步提高生肌玉红膏通过促进血管生成来提高生肌疗效,是本项目研究的重点。
第二部分制剂研究
目的：制备完成生肌玉红胶原制剂。
方法：1.分别以醇提、油提、水提法提取生肌玉红膏药物组分,进行方法对比研究。2.牛跟腱组织以酶解法提取Ⅰ型胶原。3.对Ⅰ型胶原进行肝素修饰,并通过BDC/NHS进行交联。4.修饰交联后胶原真空冷冻干燥、环氧乙烷消毒,真空包装。5.根据实验需要将生肌玉红膏在无菌环境下载入胶原。6.对胶原制剂进行电镜扫描,考察其表面及内部结构。
结果与结论：以醇提加血竭素的方法获得的减味生肌玉红膏药物组分,经预试验证实药物组分及含量符合实验要求。酶解法可有效提取Ⅰ型胶原,得率可达30%以上。真空冷冻及交联后的胶原经扫描电镜扫描显示,胶原结构更加紧密有序,呈现相互连接、平行排列的高度多孔状结构,孔径在100～200μm之间。减味生肌玉红膏载入修饰后的胶原药物呈颗粒状散布在胶原裂隙与空隙中,大小约10μm-50μm左右,胶原基本结构未发生明显变化。
第三部分生物学评价
目的：体外细胞学试验考察减味生肌玉红膏提取液和生肌玉红胶原促细胞增殖的作用和可能的细胞毒性作用,筛选减味生肌玉红膏提取液促细胞增殖的最佳浓度。体内试验考察生肌玉红膏载入胶原的组织相容性与降解性。
方法：1.采用体外培养HUVEC的方法,通过MTT法观察减味生肌玉红膏提取液对血管内皮细胞增殖作用的影响：分为空白组,减味生肌玉红膏提取液1-8组,浓度分别为50ug/ml、100ug/ml、200ug/ml、400ug/ml、0.8mg/ml、1.6mg/ml、2.4mg/ml、3.2mg/ml不同浓度组,共9组。通过OCX8法观察生肌玉红胶原制剂对血管内皮细胞增殖作用的影响：分为对照组及实验组1(胶原组)、实验组2(减味生肌玉红膏提取液1.6mg/ml)、实验组3(减味生肌玉红膏提取液1.6mg/ml+胶原)共4组。2.将生肌玉红胶原(实验组)与交联修饰后胶原(对照组)对称植入8只新西兰白兔背部皮下,分别于术后1、2、4及8周取出2组植入材料,行肉眼观察、埋植材料近旁组织切片后H-E染色显微镜下观察及对两组材料的扫描电镜(SEM)观察。
结果与结论：1.MTT法检测细胞增殖,可见50ug/ml-2.4mg/ml的生肌玉红膏提取液各个浓度的药物均可以促进细胞增殖,且以1.6mg/ml浓度组细胞增殖最强,达到28.19%,但是增加浓度达到2.4mg/ml时细胞增殖率骤降,达3.2mg/ml时,对细胞增殖起抑制作用。CKK8法检测发现生肌玉红膏提取液及生肌玉红膏载入修饰后胶原制剂,与对照组比较均可明显促进HUVEC恂增殖,以二者结合和制剂增生率最高。
2.实验组(生肌玉红胶原)在植入兔背部局部炎症反应消失较对照组(修饰后胶原)明显提前,组织相容性较同期对照组良好,可促进近旁组织毛细血管新生与胶原新生。生肌玉红膏载入胶原较Ⅰ型胶原自身组织相容性更佳,基本上发挥出生肌玉红膏及修饰后胶原各自促创面愈合的优势,与周围组织有更加良好的生物相容性。
第四部分鸡胚绒毛尿囊膜实验
目的：探查生肌玉红胶原促鸡胚尿囊膜血管新生的效果,进一步筛选减味生肌玉红膏载入修饰后胶原的最佳剂量。
方法：实验分控制组、对照组(单纯胶原)、实验组1(减味生肌玉红膏浓度0.8mg/ml载入胶原)、实验组2(减味生肌玉红膏浓度1.6mg/ml载入胶原)、实验组3(减味生肌玉红膏浓度3.2mg/ml载入胶原),各组植入7日龄鸡胚尿囊膜孵化7d后取出胶原制剂,考察鸡胚大体状况,测定植入制剂周围鸡胚尿囊膜内微血管数、尿囊膜内血红蛋白含量。
结果与结论：各实验组鸡胚尿囊膜内血管围绕胶原呈轮辐状生长,鸡胚尿囊膜包裹标本率实验组高于空白组,实验组胶原周围鸡胚尿囊膜微血管数、胶原内血红蛋白含量均高于对照组,差异有显著性意义(P<0.01)；其中仍然以实验组2(1.6mg/ml)促血管新生作用最强,尿囊膜内血红蛋白量最多(P<0.01)。提示减味生肌玉红膏提取液以1.6mg/ml的浓度载入胶原后可以促进鸡胚尿囊膜内血管新生的作用最强,应以此浓度的制剂进行后续实验。
第五部分兔背部皮下埋植实验
目的：考察生肌玉红胶原在非缺血组织中促进血管新生从而促进创伤修复的作用效果与机制。
方法：兔背部对称埋植胶原制剂,分别于术后3d,7d、14d、28d、56d取埋植的胶原制剂标本及标本周围约0.5cm范围的组织。考察埋植后动物的行为反应及局部情况,组织病理切片观察埋植物周围组织炎性反应及胶原增殖情况,通过光度计法测定胶原内血红蛋白含量以及免疫荧光与CD34染色标记法考察周围组织微血管新生情况,Western Blot法各时间点检测VEGF、ANG-1因子的表达,通过免疫组化法考察周围组织Ⅰ、Ⅲ型胶原的生长情况及Ⅰ、Ⅲ型胶原的比例。
结果与结论：埋植后动物无特殊反应,病理切片观察显示胶原周围组织随着时间推移,炎性渗出减少,肉芽组织增生,到28d时已形成成熟的纤维结缔组织。胶原内血红蛋白检测、周围组织免疫荧光与CD34血管标记检测显示生肌玉红胶原可显著促进血管新生并诱导血管长入埋植后的胶原,其作用与对照组比较有统计学差异。Western Blot法检测VEGF、ANG-1因子实验组各时间点表达均高于对照组,VEGF在术后第3d即达到峰值,而ANG-1在术后第7d达到峰值,显示出VEGF/ANG-1对血管新生不同的调节作用。而实验组生肌玉红胶原对Ⅰ型胶原的分泌与代谢影响不明显,与对照组比较无明显差异,对Ⅲ型胶原的调节作用主要出现在肉芽生长的后期阶段,可促进Ⅲ型胶原的分泌,从而降低Ⅰ、Ⅲ型胶原的比例,使得愈合更接近于正常组织愈合而对照组接近于疤痕愈合。表明在非缺血组织中,生肌玉红胶原可显著促进周围组织微血管新生,其机制之一是在不同时间点提高了VEGF/ANG-1的表达,二者的协同作用促进了血管的新生。生肌玉红胶原能调节胶原形成并调节Ⅰ、Ⅲ型胶原的比例而发挥促进组织愈合并减少疤痕愈合的作用。
第六部分大鼠后肢缺血组织埋植实验
目的：探查生肌玉红胶原在缺血组织中促进血管新生的作用效果与作用机制。
方法：制作大鼠后肢缺血模型,并于后肢缺血组织中埋植胶原制剂,分别于术后3d,7d、14d、28d取埋植的胶原制剂标本及标本周围约0.5cm范围的组织。通过激光散斑成像系统观察大鼠后肢创伤组织在各时间点的血流灌注情况,通过埋植胶原内的血红蛋白含量、周围组织各时间点的CD34微血管免疫组化标记计数观察检测生肌玉红胶原在缺血组织中对微血管新生的作用。实时荧光定量RT-PCR测定检测缺血组织各时间点HIF-1α-mRNA、VEGF-mRNA基因的表达,考察缺血组织中制剂促血管新生的作用机理。
结果与结论：激光散斑成像血流灌注检测显示术后当时大鼠后肢创伤组织呈现出明显的缺血状态,而术后3d却显示出明显的充血状态,术后7d、14d呈现出低于术前的血流灌注显示出缺血状态,至术后28d逐步回升。生肌玉红胶原仍然表现出强于胶原自身促微血管新生的作用,其作用机理是能够促进组织中HIF-1α-mRNA、VEGF-mRNA基因的高表达,其表达强度明显高于单纯埋植胶原组,目.表达作用时间较长。
Part One Background
Surgery of Traditional Chinese Medicine is also named ulcer disease at ancient times, and it has advantages in chronic ulcer of lower extremity treatment. During the treatment, TCM surgery had accumulated rich clinical experiences. Shengji Yuhong Ointment is a representative drug for chronic lower extremity ulcers in III-IV period, to make the wound granulation fresh and the size decreased. Previous clinical study of Shengji Yuhong Ointment shows that the Shengji Yuhong Ointment can promote microcirculation, accelerate growth of granulation and epithelial, which lead to healing of chronic lower extremity ulcer wound.
Many studies tried to improve the curative effect by adjusting the composition of Shengji Yuhong Ointment. Therefore, how to optimize the composition becomes one of the hotspots of the Surgery of Traditional Chinese Medicine. This study is going to remove the Calomelas from original Shengji Yuhong Ointment in order to reduce its toxicity and test its impact on angiogenesis.
High level, rapid vascularization is a basic issue of wound healing research. Use collagen as holder for growth factor to build a biological material which can promote vessel growth and can be implanted into body to promote angiogenesis, has become the forefront and hotspot of international researches. Whether the combination of Shengji Yuhong Ointment and modified collagen can enhance curative effect by promoting angiogenesis, is the focus of this project.
Part Two Preparation
Objective:Load Shengji Yuhong Ointment to the modified collagen
Method:1.Use alcohol extraction, oil extraction and water extraction metod, to extract the components of Shengji Yuhong Ointment. Compare those metods.
2. Use enzymatic extraction to extract type1collagen from bovine Achilles tendon organizations
3. Modified the type I collagen by heparin method, and cross-linked by EDC/NHS.
4. Vacuum freeze-dried the modified cross-linked collagen, and ethylene oxide sterilized them.。
5. According to the requirements of experiment, the Shengji Yuhong Ointment should be loaded in the sterile environment into collagen.
6. Scanning the collagen mixture by electron microscopy to study the surface and internal structure.
Result and Conclusion:The modified Shengji Yuhong extraction extracted by alcohol method and added in dracorhodin is testified by preliminary test, that the component and concentration is within the experimental requirements. The enzymatic method can effectively extract the type I collagen, and the success rate up to30%. Scanning by electron microscopy, the vacuum freezed and cross-linking modified collagen shows that the collagen structure more closely and orderly which has interconnected, parallel arranged-highly porous structure and the pore size between100to200μm. The component of Shengji Yuhong Ointment is granular spreading in the collagen cracks and gaps, with the size of about10μm to50μm. The basic structure of collagen did not change significantly.
Part Three Biological Evaluation
Purpose:Observe the function of Shengji Yuhong extraction and ShengJi Yuhong Ointment loaded by collagen about cell proliferation promotion and cytotoxicity by vitro cytology test. Find out the concentration of modified Shengji Yuhong Ointment extraction which can promote cell proliferation best. To examine the histocompatibility of collagen loaded by Shengji Yuhong Ointment by experiments in vivo.
Method:1. Use in vitro HUVEC method, observe the effect of Shengji Yuhong alcohol extracts on vascular endothelial cell proliferation by MTT test:set up control group and Shengji Yuhong Ointment alcohol extracts1to8groups, the concentration respectively is50ug/ml,100ug/ml,200ug/ml,400ug/ml,0.8mg/ml,1.6mg/ml,2.4mg/ml,3.2mg/ml, total group amount is nine. Observe the effect of Shengji Yuhong Ointment collagen on vascular endothelial cell proliferation by CCK-8test: set up control group and experimental group1(collagen group), experimental group2(modified Shengji Yuhong Ointment extract of1.6mg/ml), experimental group3(collagen+modified Shengji Yuhong Ointment extracts1.6mg/ml), there are three groups.
Implanted Shengji Yuhong Ointment collagen (experimental group) and collagen with cross-linking modification (control group) into eight New Zealand rabbits dorsal subcutaneous tissue. Take out the material of two groups at postoperative1,2,4and8weeks respectively, observe them by visual, then observe the tissue around the material under microscope after histotomy and H-E staining, and observe material from two groups by electron microscopy (SEM) observation.
Results and Conclusions:1.MTT test to detect cell proliferation, it shows the Shengji Yuhong alcohol extracts concentrations from50ug/ml to2.4mg/ml can promote cell proliferation, and the concentration at1.6mg/ml has the highest cell proliferation rate which can reach28.19%; when reaches2.4mg/ml concentration the proliferation rates plummets.; when reaches3.2mg/ml, it even can play a disincentive effect on cell proliferation. Compared with the control group through CKK-8, modified Shengji Yuhong Ointment extracts and modified collagen loaded by Shengji Yuhong Ointment can significantly promote the proliferation of HUVEC, and proliferation rate of Shengji Yuhong Ointment loaded into modified collagen is the highest.
2. after implantation in vivo, the Experimental group (Shengji Yuhong collagen) inflammatory reaction disappears significantly ahead than the control group (modified collagen), and has better histocompatibility than the control group at same period, and promote angiogenesis of tissues nearby and collagen neogenesis. Modified collagen loaded by Shengji Yuhong Ointment has better histocompatibility compare to type I collagen, it makes the most of the advantage of Shengji Yuhong Ointment and modified collagen respectively in wound healing, and has better biocompatibility with the surrounding tissue.
Part Four Chick chorioallantoic membrane test
Purpose:Explore the angiogenisis effect of modified collagen loaded by Shengji Yuhong Ointment, to make sure the best dose of Shengji Yuhong Ointment loaded into modified collagen.
Method:The experiment group was divided into control group, control group (collagen), experimental group1(modified Shengji Yuhong Ointment with concentration0.8mg/ml loaded into collagen), experimental group2(modified Shengji Yuhong Ointment with concentration1.6mg/ml loaded into collagen), experimental group3(modified Shengji Yuhong Ointment with concentration3.2mg/ml loaded collagen), each group was implanted into7-day-old chick embryo chorioallantoic membrane and remove the collagen after7days incubation, to study the general situation of chick embryo, measure the amounts of microvessel which around the implanted drugs in the chicken embryo allantoic membrane, and measure the hemoglobin content of collagen in collagen.
Results and conclusions:blood vessels in chorioallantoic membrane of experiment groups were spoke-like growth and the chorioallantoic membrane wrapped specimens rate of experiment groups is higher than other groups, microvessel counts of the chick embryo chorioallantoic membrane and collagen hemoglobin content of experimental group are higher than control group, the difference was significant (P<0.01); still the group(1.6mg/ml concentration) has strongest angiogenesis function and with the most hemoglobin in chorioallantoic membrane (P<0.01). It implies that modified Shengji Yuhong extracts with1.6mg/ml concentration loaded into modified collagen has the strongest effect on promoting chick embryo chorioallantoic membrane angiogenesis, and as a result this concentration should be selected for subsequent experiments.
Part Five Underneath implant experiment on rabbit dorsal
Objective:To find out the effect of modified collagen loaded by Shengji Yuhong Ointment on promoting non-ischemic tissue angiogenesis.
Method:Symmetrical implant collagen into rabbit back, take out implanted collagen preparations specimens and specimen about5cm around the range of organizations respectively after3d,7d,14d,28d,56d. Observe the animal's behavior, response and local situation after the implant. Observe the inflammatory reaction and collagen proliferation around the implant by histopathological section, measure the hemoglobin content by photometric method, study angiogenesis by immunofluorescence and CD34staining notation, measure the VEGF, ANG-1cytokine expression at different time by Western Blot, observe the growth and proportion of type Ⅰ, Ⅲ collagen in the surrounding tissue by immunohistochemical method.
Results and Conclusion:Animals have no special response after implanted, the pathological section shows as time goes by, the inflammatory exudate reduces, granulation tissue grows and the mature fibrous connective tissue formed at28th days. Hemoglobin test of collagen, immunofluorescence and CD34vascular markers test of surrounding tissue shows Shengji Yuhong collagen can significantly promote angiogenesis and induce vessels grow into collagen after implanted, it has statistical differences compared to control group. Western Blot shows that the VEGF and ANG-1factor expression of experimental group is higher than control group. VEGF expression reached its peak at3rd day while ANG-1reached its peak at the7th day, it shows different action of VEGF/ANG-1in regulating angiogenesis. Shengji Yuhong collagen from experimental group has no significant effect on secretion and metabolic of type I collagen, which has no significant difference compared to control group; the regulation on collagen type Ⅲ occurs in the later period of granulation growth, and it promotes the secretion of collagen type Ⅲ to reduce the proportion of collagen type I and III which makes healing more close to the normal, and the tissue healing in the control group is more likely the scar healing. It implies that in non-ischemic tissue Shengji Yuhong collagen can significantly promote the angiogenesis, one of its mechanisms is enhance the expression of VEGF/ANG-1at different time, and the synergy can promote angiogenesis. Shengji Yuhong collagen can coordinated regulation collagen formation and adjustment proportion of Ⅰ, Ⅲ type collagen to promote tissue healing and reduce scar healing.
Part Six implant experiment on ischemic tissue of rats'hindlimbs
Objective:To find out the angiogenic effect of modified collagen loaded by Shengji Yuhong Ointment in ischemic tissue.
Methods:Make the rats'ischemic hindlimbs model, implanted collagen in ischemic tissue of rats'hindlimbs, take out implanted collagen preparations specimens and specimen about5cm around the range of organizations respectively after3d,7d,14d,28d; observed blood perfusion of ischemic tissue from Hindlimb by laser speckle imaging system, measure the hemoglobin content and CD34microvascular immunohistochemical markers count to observe the function of Shengji Yuhong collagen in micrangium-genesis. Use real-time fluorescent quantitative RT-PCR to detect the gene expression of HIF-1α mRNA and VEGF-mRNA in ischemic tissue at different time points, and study the mechanism of the agents in promoting angiogenesis in ischemic tissue.
Results and Conclusion:Laser speckle imaging perfusion test shows that postoperative hindlimb tissue was in ischemic situation, but it changed to obvious congestion situation at postoperative3rd day; postoperative7th day and14th day has a lower bloodflow perfusion than the preoperative shows it in ischemia situation and it gradually return to former condition at postoperative28th day. Shengji Yuhong collagen still shows stronger function than collagen in micrangium-genesis, the mechanism is to promote high gene expression of HIF-1α-mRNA and VEGF-mRNA. The expression intensity of Shengji Yuhong collagen is significantly higher than the collagen group, and it has longer action time.
中医外科古代又名疡科,下肢慢性溃疡是中医外科治疗的优势病种之一,中医在此病的治疗中积累了丰富的临床经验。对于Ⅲ-Ⅳ期慢性下肢溃疡临床多运用生肌玉红膏为代表的药物外用,使得疮面肉芽新鲜,疮面减小。既往我们对生肌玉红膏治疗慢性溃疡进行了临床研究,证明生肌玉红膏可以促进慢性下肢溃疡创面微循环、加速创面肉芽生长及上皮生长,从而促进慢性下肢溃疡创面愈合。
许多研究都在通过调整生肌玉红膏的药物组成来提高疗效。因此,如何通过进一步改良而提高疗效,是中医外科研究的热点之一。本研究拟用生肌玉红膏原方去除轻粉,以减轻其毒性,检测其对血管新生与发生的影响。
高水平、快速的血管化一直是慢性创面修复研究的一个基本课题。应用胶原作为支架载入生长因子制造促血管生长的生物材料植入机体促血管生长为目前国际研究开发的前沿与热点之一。将修饰后的胶原作为减味生肌玉红膏敷料载体,发挥两者促创面愈合优势,是否可以进一步提高生肌玉红膏通过促进血管生成来提高生肌疗效,是本项目研究的重点。
第二部分制剂研究
目的：制备完成生肌玉红胶原制剂。
方法：1.分别以醇提、油提、水提法提取生肌玉红膏药物组分,进行方法对比研究。2.牛跟腱组织以酶解法提取Ⅰ型胶原。3.对Ⅰ型胶原进行肝素修饰,并通过BDC/NHS进行交联。4.修饰交联后胶原真空冷冻干燥、环氧乙烷消毒,真空包装。5.根据实验需要将生肌玉红膏在无菌环境下载入胶原。6.对胶原制剂进行电镜扫描,考察其表面及内部结构。
结果与结论：以醇提加血竭素的方法获得的减味生肌玉红膏药物组分,经预试验证实药物组分及含量符合实验要求。酶解法可有效提取Ⅰ型胶原,得率可达30%以上。真空冷冻及交联后的胶原经扫描电镜扫描显示,胶原结构更加紧密有序,呈现相互连接、平行排列的高度多孔状结构,孔径在100～200μm之间。减味生肌玉红膏载入修饰后的胶原药物呈颗粒状散布在胶原裂隙与空隙中,大小约10μm-50μm左右,胶原基本结构未发生明显变化。
第三部分生物学评价
目的：体外细胞学试验考察减味生肌玉红膏提取液和生肌玉红胶原促细胞增殖的作用和可能的细胞毒性作用,筛选减味生肌玉红膏提取液促细胞增殖的最佳浓度。体内试验考察生肌玉红膏载入胶原的组织相容性与降解性。
方法：1.采用体外培养HUVEC的方法,通过MTT法观察减味生肌玉红膏提取液对血管内皮细胞增殖作用的影响：分为空白组,减味生肌玉红膏提取液1-8组,浓度分别为50ug/ml、100ug/ml、200ug/ml、400ug/ml、0.8mg/ml、1.6mg/ml、2.4mg/ml、3.2mg/ml不同浓度组,共9组。通过OCX8法观察生肌玉红胶原制剂对血管内皮细胞增殖作用的影响：分为对照组及实验组1(胶原组)、实验组2(减味生肌玉红膏提取液1.6mg/ml)、实验组3(减味生肌玉红膏提取液1.6mg/ml+胶原)共4组。2.将生肌玉红胶原(实验组)与交联修饰后胶原(对照组)对称植入8只新西兰白兔背部皮下,分别于术后1、2、4及8周取出2组植入材料,行肉眼观察、埋植材料近旁组织切片后H-E染色显微镜下观察及对两组材料的扫描电镜(SEM)观察。
结果与结论：1.MTT法检测细胞增殖,可见50ug/ml-2.4mg/ml的生肌玉红膏提取液各个浓度的药物均可以促进细胞增殖,且以1.6mg/ml浓度组细胞增殖最强,达到28.19%,但是增加浓度达到2.4mg/ml时细胞增殖率骤降,达3.2mg/ml时,对细胞增殖起抑制作用。CKK8法检测发现生肌玉红膏提取液及生肌玉红膏载入修饰后胶原制剂,与对照组比较均可明显促进HUVEC恂增殖,以二者结合和制剂增生率最高。
2.实验组(生肌玉红胶原)在植入兔背部局部炎症反应消失较对照组(修饰后胶原)明显提前,组织相容性较同期对照组良好,可促进近旁组织毛细血管新生与胶原新生。生肌玉红膏载入胶原较Ⅰ型胶原自身组织相容性更佳,基本上发挥出生肌玉红膏及修饰后胶原各自促创面愈合的优势,与周围组织有更加良好的生物相容性。
第四部分鸡胚绒毛尿囊膜实验
目的：探查生肌玉红胶原促鸡胚尿囊膜血管新生的效果,进一步筛选减味生肌玉红膏载入修饰后胶原的最佳剂量。
方法：实验分控制组、对照组(单纯胶原)、实验组1(减味生肌玉红膏浓度0.8mg/ml载入胶原)、实验组2(减味生肌玉红膏浓度1.6mg/ml载入胶原)、实验组3(减味生肌玉红膏浓度3.2mg/ml载入胶原),各组植入7日龄鸡胚尿囊膜孵化7d后取出胶原制剂,考察鸡胚大体状况,测定植入制剂周围鸡胚尿囊膜内微血管数、尿囊膜内血红蛋白含量。
结果与结论：各实验组鸡胚尿囊膜内血管围绕胶原呈轮辐状生长,鸡胚尿囊膜包裹标本率实验组高于空白组,实验组胶原周围鸡胚尿囊膜微血管数、胶原内血红蛋白含量均高于对照组,差异有显著性意义(P<0.01)；其中仍然以实验组2(1.6mg/ml)促血管新生作用最强,尿囊膜内血红蛋白量最多(P<0.01)。提示减味生肌玉红膏提取液以1.6mg/ml的浓度载入胶原后可以促进鸡胚尿囊膜内血管新生的作用最强,应以此浓度的制剂进行后续实验。
第五部分兔背部皮下埋植实验
目的：考察生肌玉红胶原在非缺血组织中促进血管新生从而促进创伤修复的作用效果与机制。
方法：兔背部对称埋植胶原制剂,分别于术后3d,7d、14d、28d、56d取埋植的胶原制剂标本及标本周围约0.5cm范围的组织。考察埋植后动物的行为反应及局部情况,组织病理切片观察埋植物周围组织炎性反应及胶原增殖情况,通过光度计法测定胶原内血红蛋白含量以及免疫荧光与CD34染色标记法考察周围组织微血管新生情况,Western Blot法各时间点检测VEGF、ANG-1因子的表达,通过免疫组化法考察周围组织Ⅰ、Ⅲ型胶原的生长情况及Ⅰ、Ⅲ型胶原的比例。
结果与结论：埋植后动物无特殊反应,病理切片观察显示胶原周围组织随着时间推移,炎性渗出减少,肉芽组织增生,到28d时已形成成熟的纤维结缔组织。胶原内血红蛋白检测、周围组织免疫荧光与CD34血管标记检测显示生肌玉红胶原可显著促进血管新生并诱导血管长入埋植后的胶原,其作用与对照组比较有统计学差异。Western Blot法检测VEGF、ANG-1因子实验组各时间点表达均高于对照组,VEGF在术后第3d即达到峰值,而ANG-1在术后第7d达到峰值,显示出VEGF/ANG-1对血管新生不同的调节作用。而实验组生肌玉红胶原对Ⅰ型胶原的分泌与代谢影响不明显,与对照组比较无明显差异,对Ⅲ型胶原的调节作用主要出现在肉芽生长的后期阶段,可促进Ⅲ型胶原的分泌,从而降低Ⅰ、Ⅲ型胶原的比例,使得愈合更接近于正常组织愈合而对照组接近于疤痕愈合。表明在非缺血组织中,生肌玉红胶原可显著促进周围组织微血管新生,其机制之一是在不同时间点提高了VEGF/ANG-1的表达,二者的协同作用促进了血管的新生。生肌玉红胶原能调节胶原形成并调节Ⅰ、Ⅲ型胶原的比例而发挥促进组织愈合并减少疤痕愈合的作用。
第六部分大鼠后肢缺血组织埋植实验
目的：探查生肌玉红胶原在缺血组织中促进血管新生的作用效果与作用机制。
方法：制作大鼠后肢缺血模型,并于后肢缺血组织中埋植胶原制剂,分别于术后3d,7d、14d、28d取埋植的胶原制剂标本及标本周围约0.5cm范围的组织。通过激光散斑成像系统观察大鼠后肢创伤组织在各时间点的血流灌注情况,通过埋植胶原内的血红蛋白含量、周围组织各时间点的CD34微血管免疫组化标记计数观察检测生肌玉红胶原在缺血组织中对微血管新生的作用。实时荧光定量RT-PCR测定检测缺血组织各时间点HIF-1α-mRNA、VEGF-mRNA基因的表达,考察缺血组织中制剂促血管新生的作用机理。
结果与结论：激光散斑成像血流灌注检测显示术后当时大鼠后肢创伤组织呈现出明显的缺血状态,而术后3d却显示出明显的充血状态,术后7d、14d呈现出低于术前的血流灌注显示出缺血状态,至术后28d逐步回升。生肌玉红胶原仍然表现出强于胶原自身促微血管新生的作用,其作用机理是能够促进组织中HIF-1α-mRNA、VEGF-mRNA基因的高表达,其表达强度明显高于单纯埋植胶原组,目.表达作用时间较长。
Part One Background
Surgery of Traditional Chinese Medicine is also named ulcer disease at ancient times, and it has advantages in chronic ulcer of lower extremity treatment. During the treatment, TCM surgery had accumulated rich clinical experiences. Shengji Yuhong Ointment is a representative drug for chronic lower extremity ulcers in III-IV period, to make the wound granulation fresh and the size decreased. Previous clinical study of Shengji Yuhong Ointment shows that the Shengji Yuhong Ointment can promote microcirculation, accelerate growth of granulation and epithelial, which lead to healing of chronic lower extremity ulcer wound.
Many studies tried to improve the curative effect by adjusting the composition of Shengji Yuhong Ointment. Therefore, how to optimize the composition becomes one of the hotspots of the Surgery of Traditional Chinese Medicine. This study is going to remove the Calomelas from original Shengji Yuhong Ointment in order to reduce its toxicity and test its impact on angiogenesis.
High level, rapid vascularization is a basic issue of wound healing research. Use collagen as holder for growth factor to build a biological material which can promote vessel growth and can be implanted into body to promote angiogenesis, has become the forefront and hotspot of international researches. Whether the combination of Shengji Yuhong Ointment and modified collagen can enhance curative effect by promoting angiogenesis, is the focus of this project.
Part Two Preparation
Objective:Load Shengji Yuhong Ointment to the modified collagen
Method:1.Use alcohol extraction, oil extraction and water extraction metod, to extract the components of Shengji Yuhong Ointment. Compare those metods.
2. Use enzymatic extraction to extract type1collagen from bovine Achilles tendon organizations
3. Modified the type I collagen by heparin method, and cross-linked by EDC/NHS.
4. Vacuum freeze-dried the modified cross-linked collagen, and ethylene oxide sterilized them.。
5. According to the requirements of experiment, the Shengji Yuhong Ointment should be loaded in the sterile environment into collagen.
6. Scanning the collagen mixture by electron microscopy to study the surface and internal structure.
Result and Conclusion:The modified Shengji Yuhong extraction extracted by alcohol method and added in dracorhodin is testified by preliminary test, that the component and concentration is within the experimental requirements. The enzymatic method can effectively extract the type I collagen, and the success rate up to30%. Scanning by electron microscopy, the vacuum freezed and cross-linking modified collagen shows that the collagen structure more closely and orderly which has interconnected, parallel arranged-highly porous structure and the pore size between100to200μm. The component of Shengji Yuhong Ointment is granular spreading in the collagen cracks and gaps, with the size of about10μm to50μm. The basic structure of collagen did not change significantly.
Part Three Biological Evaluation
Purpose:Observe the function of Shengji Yuhong extraction and ShengJi Yuhong Ointment loaded by collagen about cell proliferation promotion and cytotoxicity by vitro cytology test. Find out the concentration of modified Shengji Yuhong Ointment extraction which can promote cell proliferation best. To examine the histocompatibility of collagen loaded by Shengji Yuhong Ointment by experiments in vivo.
Method:1. Use in vitro HUVEC method, observe the effect of Shengji Yuhong alcohol extracts on vascular endothelial cell proliferation by MTT test:set up control group and Shengji Yuhong Ointment alcohol extracts1to8groups, the concentration respectively is50ug/ml,100ug/ml,200ug/ml,400ug/ml,0.8mg/ml,1.6mg/ml,2.4mg/ml,3.2mg/ml, total group amount is nine. Observe the effect of Shengji Yuhong Ointment collagen on vascular endothelial cell proliferation by CCK-8test: set up control group and experimental group1(collagen group), experimental group2(modified Shengji Yuhong Ointment extract of1.6mg/ml), experimental group3(collagen+modified Shengji Yuhong Ointment extracts1.6mg/ml), there are three groups.
Implanted Shengji Yuhong Ointment collagen (experimental group) and collagen with cross-linking modification (control group) into eight New Zealand rabbits dorsal subcutaneous tissue. Take out the material of two groups at postoperative1,2,4and8weeks respectively, observe them by visual, then observe the tissue around the material under microscope after histotomy and H-E staining, and observe material from two groups by electron microscopy (SEM) observation.
Results and Conclusions:1.MTT test to detect cell proliferation, it shows the Shengji Yuhong alcohol extracts concentrations from50ug/ml to2.4mg/ml can promote cell proliferation, and the concentration at1.6mg/ml has the highest cell proliferation rate which can reach28.19%; when reaches2.4mg/ml concentration the proliferation rates plummets.; when reaches3.2mg/ml, it even can play a disincentive effect on cell proliferation. Compared with the control group through CKK-8, modified Shengji Yuhong Ointment extracts and modified collagen loaded by Shengji Yuhong Ointment can significantly promote the proliferation of HUVEC, and proliferation rate of Shengji Yuhong Ointment loaded into modified collagen is the highest.
2. after implantation in vivo, the Experimental group (Shengji Yuhong collagen) inflammatory reaction disappears significantly ahead than the control group (modified collagen), and has better histocompatibility than the control group at same period, and promote angiogenesis of tissues nearby and collagen neogenesis. Modified collagen loaded by Shengji Yuhong Ointment has better histocompatibility compare to type I collagen, it makes the most of the advantage of Shengji Yuhong Ointment and modified collagen respectively in wound healing, and has better biocompatibility with the surrounding tissue.
Part Four Chick chorioallantoic membrane test
Purpose:Explore the angiogenisis effect of modified collagen loaded by Shengji Yuhong Ointment, to make sure the best dose of Shengji Yuhong Ointment loaded into modified collagen.
Method:The experiment group was divided into control group, control group (collagen), experimental group1(modified Shengji Yuhong Ointment with concentration0.8mg/ml loaded into collagen), experimental group2(modified Shengji Yuhong Ointment with concentration1.6mg/ml loaded into collagen), experimental group3(modified Shengji Yuhong Ointment with concentration3.2mg/ml loaded collagen), each group was implanted into7-day-old chick embryo chorioallantoic membrane and remove the collagen after7days incubation, to study the general situation of chick embryo, measure the amounts of microvessel which around the implanted drugs in the chicken embryo allantoic membrane, and measure the hemoglobin content of collagen in collagen.
Results and conclusions:blood vessels in chorioallantoic membrane of experiment groups were spoke-like growth and the chorioallantoic membrane wrapped specimens rate of experiment groups is higher than other groups, microvessel counts of the chick embryo chorioallantoic membrane and collagen hemoglobin content of experimental group are higher than control group, the difference was significant (P<0.01); still the group(1.6mg/ml concentration) has strongest angiogenesis function and with the most hemoglobin in chorioallantoic membrane (P<0.01). It implies that modified Shengji Yuhong extracts with1.6mg/ml concentration loaded into modified collagen has the strongest effect on promoting chick embryo chorioallantoic membrane angiogenesis, and as a result this concentration should be selected for subsequent experiments.
Part Five Underneath implant experiment on rabbit dorsal
Objective:To find out the effect of modified collagen loaded by Shengji Yuhong Ointment on promoting non-ischemic tissue angiogenesis.
Method:Symmetrical implant collagen into rabbit back, take out implanted collagen preparations specimens and specimen about5cm around the range of organizations respectively after3d,7d,14d,28d,56d. Observe the animal's behavior, response and local situation after the implant. Observe the inflammatory reaction and collagen proliferation around the implant by histopathological section, measure the hemoglobin content by photometric method, study angiogenesis by immunofluorescence and CD34staining notation, measure the VEGF, ANG-1cytokine expression at different time by Western Blot, observe the growth and proportion of type Ⅰ, Ⅲ collagen in the surrounding tissue by immunohistochemical method.
Results and Conclusion:Animals have no special response after implanted, the pathological section shows as time goes by, the inflammatory exudate reduces, granulation tissue grows and the mature fibrous connective tissue formed at28th days. Hemoglobin test of collagen, immunofluorescence and CD34vascular markers test of surrounding tissue shows Shengji Yuhong collagen can significantly promote angiogenesis and induce vessels grow into collagen after implanted, it has statistical differences compared to control group. Western Blot shows that the VEGF and ANG-1factor expression of experimental group is higher than control group. VEGF expression reached its peak at3rd day while ANG-1reached its peak at the7th day, it shows different action of VEGF/ANG-1in regulating angiogenesis. Shengji Yuhong collagen from experimental group has no significant effect on secretion and metabolic of type I collagen, which has no significant difference compared to control group; the regulation on collagen type Ⅲ occurs in the later period of granulation growth, and it promotes the secretion of collagen type Ⅲ to reduce the proportion of collagen type I and III which makes healing more close to the normal, and the tissue healing in the control group is more likely the scar healing. It implies that in non-ischemic tissue Shengji Yuhong collagen can significantly promote the angiogenesis, one of its mechanisms is enhance the expression of VEGF/ANG-1at different time, and the synergy can promote angiogenesis. Shengji Yuhong collagen can coordinated regulation collagen formation and adjustment proportion of Ⅰ, Ⅲ type collagen to promote tissue healing and reduce scar healing.
Part Six implant experiment on ischemic tissue of rats'hindlimbs
Objective:To find out the angiogenic effect of modified collagen loaded by Shengji Yuhong Ointment in ischemic tissue.
Methods:Make the rats'ischemic hindlimbs model, implanted collagen in ischemic tissue of rats'hindlimbs, take out implanted collagen preparations specimens and specimen about5cm around the range of organizations respectively after3d,7d,14d,28d; observed blood perfusion of ischemic tissue from Hindlimb by laser speckle imaging system, measure the hemoglobin content and CD34microvascular immunohistochemical markers count to observe the function of Shengji Yuhong collagen in micrangium-genesis. Use real-time fluorescent quantitative RT-PCR to detect the gene expression of HIF-1α mRNA and VEGF-mRNA in ischemic tissue at different time points, and study the mechanism of the agents in promoting angiogenesis in ischemic tissue.
Results and Conclusion:Laser speckle imaging perfusion test shows that postoperative hindlimb tissue was in ischemic situation, but it changed to obvious congestion situation at postoperative3rd day; postoperative7th day and14th day has a lower bloodflow perfusion than the preoperative shows it in ischemia situation and it gradually return to former condition at postoperative28th day. Shengji Yuhong collagen still shows stronger function than collagen in micrangium-genesis, the mechanism is to promote high gene expression of HIF-1α-mRNA and VEGF-mRNA. The expression intensity of Shengji Yuhong collagen is significantly higher than the collagen group, and it has longer action time.
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