The Experimental Research On Angiogenesis By Sheng Ji Yu Hong Loading On Modified Collagen Matrices

Part One BackgroundSurgery 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 PreparationObjective:Load Shengji Yuhong Ointment to the modified collagenMethod: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 EvaluationPurpose: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 testPurpose: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 dorsalObjective: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'hindlimbsObjective: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.