| Objectives:At present based on the fact that soft tissue injuries of each part of the body caused by such factors as trauma, tumors or congenital malformations which have serious effects on both patients’ appearance and mentality, the reparation and reconstruction of soft tissue injuries is still a big challenge faced by plastic surgery. Adipose transplantation technique, especially autologous adipose transplantation is one of the most commonly-used method of tissue reparation and reconstruction. Autologous adipose tissues are characterized by rich source, convenience, safety and no immune rejection response. However, the survival rate of transplanted adipose tissue is usually between 30% and 60%, which is rather low and affects greatly the long-term therapy of autologous adipose transplantation. The insufficient establishment of blood supply in transplantation area especially that transplantation tissues in central areas can’t get enough oxygen and nutrition, influence the long-term survival rate of transplanted adipose tissues. As a result, how to promote the reconstruction of internal blood supply of transplanted adipose tissues, shorten low blood period and increase the survival rate of transplanted adipose tissues become a hot and difficult point in the research of autologous adipose transplantation. The main observed indicators in research on the effects of recombinant human erythropoietin on survival rate of adipose transplantation are microvascular generation condition of transplanted adipose tissues. morphological structures of adipose tissues and cells, the percentage of necrosis area in each section sight, the quality and volume changes of changes of adipose tissues, and recombinant human erythropoietin can be produced industrially on a large scale by genetic engineering, It is easily acquired and convenience to be applied into clinic. Bone marrow mesenchymal stem cells can be induced to differentiate into adipose cells and themselves can secrete some cytokines involved in vascular remodeling under certain conditions, their obvervational indexes that effect on adipose transplantation are in accord with recombinant human erythropoietin. This topic is mainly to study whether recombinant human erythropoietin and bone marrow mesenchymal stem cells play a positive role in the survival rate of adipose transplantation,which can provide some thinking and application room for clinical experiences of adipose transplantation.Methods:1. Selection of experimental animal:72 health Balb/c mice are chosen as experimental animals. Each which weighs 20±1.5g is seven weeks old. all is female. They were bought from experimental animals center of Kunming medical university.2. Establishment of animal model:adipose tissues are excised from thirty-six Balb/c mice and divided into thirty-six equal parts, each weighs 0.3g. Another 36 Balb/c mice are divided into group A, B, C, each group includes 12 mice.They are anesthetized by pentobarbital. When the anesthesia works, the right back of mice are cut an 0.3cm incision by ophthalmic scissor. The 0.3g adipose tissue which is fixed of 3000IU/kg rHuEPO is injected into right back of mice of group A. The 0.3g adipose tissue which is fixed of 5x105 mice’s bone marrow mesenchymal stem cells is injected into right back of mice of group B.The 0.3g adipose tissue is injected into right back of mice of group C. The incision is sutured after adipose injection.3. Grouping and intervention:The rhEPO is injected into adipose tissue every three days, a total of three times. Group B (stem cells group):A total of 12 mice, clean water and normal food are gave. Group C (blank group):A total of 12 mice, clean water and normal food are gave.4. Draw materials:12 mice are decapitated on 30th,60th and 90th day after operation, four of each group will be decapitated. The adipose tissues along with envelope are removed completely. Using electronic scales to weigh transplanted adipose tissues. Using drainage method to measure their volumes. Respectively 12 adipose masses are kept in 4% paraformaldehyde solution for 24h. Dehydration, paraffin embedding, sectioning(3um), the tissues are routinely stained by H&E. Using 100 magnification microscope to observe organization and cells morphology. Select all the successive non-overlapping fields of view along any axis under 100 magnification, the percentage of necrosis area is calculated by point-counting. Select five successive non-overlapping fields of view along any axis and count microvessels quantity after immunohistochemical staining under 200 magnification,and calculate the average.Results:1.The wet weights of transplanted adipose tissues of group A and group B are greater than the group C’s at the same time point on 30th,60thand 90th day after surgery, and the difference is statistically significant (P<0.05). The wet weights of each group show a trend of decrease. The wet weights of adipose tissues of group A and group B’s are near, and the difference is not statistically significant (P>0.05). The difference of compare within group is statistically significant at different time points (P<0.05)2. The volumes of transplanted adipose tissues of group A and group B are greater than the group C’s at the same time point on 30th,60thand 90th day after surgery, and the difference is statistically significant (P<0.05). The volumes of each group decrease over time. The volumes of adipose tissues of group A and group B are near, and the difference is not statistically significant (P>0.05). The difference of compare within group is statistically significant at different time points (P<0.05).3.The H&E staining slices are observed on 30th,60thand 90th day after surgery. Early adipose tissues of each group show that cells structures are complete and clear, cells differentiations are mature, cells arrangements are regular. Each group appears different degrees of necrotic tissues, cavitation number increases over time. Group C is particularly serious. The percentages of necrosis area of transplanted adipose tissues of group A and group B are smaller than the group C’s at the same time point. The percentages of necrosis area of each group show a trend of increase. The percentages of necrosis area of adipose tissues of group A and group B are near, and there is not statistically significant difference (P>0.05). The difference of compare within group is statistically significant at different time points (P<0.05)4. Calculate the quantity of microvessels marked by positive cells of CD31 protein’s expression under light microscope. The microvessels quantity of group A and group B are greater than the group C’s at the same time point. The microvessels quantity of each group shows a trend of increase. The microvessels quantity of adipose tissues of group A and group B are near, and there is not statistically significant difference (P>0.05). Intra-group comparison:the difference is statistically significant at different time points (P<0.05)Conclusions:1. Balb/c mice, which can simulate experimental process of autologous adipose transplantation and possess advantage of simple resource, low cost and good experimental repeatability, is a good animal modle to study autologous adipose transplantation.2. Both recombinant human erythropoietin and bone marrow mesenchymal stem cells can increase the survival rate of transplanted adipose tissues.3. Both recombinant human erythropoietin and bone marrow mesenchymal stem cells can promote angiogenesis of transplanted adipose tissues. |
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