三种可注射水凝胶促进大鼠缺血下肢微血管再生对比研究

Comparative study of three kinds of injectable hydrogels promoting angiogenesis in rats with ischemic limbs

目的通过制备PLGA-PEG-PLGA温敏性水凝胶、海藻酸钠-壳聚糖复合水凝胶和slant自组装多肽水凝胶，探讨大鼠下肢肌肉注射水凝胶对微血管再生的作用，为应用水凝胶材料治疗缺血性疾病提供新的策略和理论依据。方法制备出可注射型PLGA-PEG-PLGA温敏性水凝胶、海藻酸钠-壳聚糖温敏复合水凝胶和slanc自组装多肽水凝胶。SD雄性大鼠随机分为PLGA-PEG-PLGA组（PPP组）、海藻酸钠-壳聚糖组（SA-CS组）、slanc组、PBS对照组及正常对照组。前四组建立雄性大鼠急性下肢缺血模型，缺血24h后，下肢肌肉多点注射1．0ml上述四种溶液；正常对照组行假手术处理。分别于4周、8周后，取各组大鼠下肢腓肠肌做组织切片。免疫组化检测CD31，检测各组大鼠的微血管密度，评价三种水凝胶对大鼠下肢微血管再生作用效果。结果各组内比较，8周时，三种水凝胶组大鼠血管密度比4周时有所增加（P〈0．05）。4周时，与PBS对照组比较，PPP组微血管密度未见明显增加（8．67±1．66比7．33±1．66），P〉0．05），SA-CS组（15．33±3．64）及slanc组（13．78±2．99）微血管密度增加（P〈0．05）。8周时，与PBS对照组（8．72±1．36）比较，PPP组（14．39±1．33）、SA-CS组（23．33±2．11）及slanc组（18．61±1，72）微血管密度明显增加（P〈0．05），其中SA-CS组微血管密度增加最多（P〈0．05）。结论设计合成的三种水凝胶都具有一定促进血管再生的能力，其中海藻酸钠-壳聚糖复合水凝胶促进血管再生功能最为明显，在组织工程修复、器官组织再生、缺血性疾病治疗方面有潜在临床应用价值。

Objective To investigate the effects of thermosensitive hydrogel PLGA-PEG-FLGA, composite hydrogel with alginate and chitosan, self-assembled peptide hydrogel slane on the microvascular regenerationand then provide the new strategies and theory basis for patients with ischemic diseases. Methods Male SD rats were randomly divided into PLGA-PEG-PLGA group （PPP group）, alginate-chitosan group （SA-CS group ）, slanc group, PBS control group and normal control group. Acute hind limb ischemia models were established and 24 hours after ischemia, 1.0 ml of the four solutions were injected multi-point in limb muscle respectively, normal control group underwent sham operation, After 4 weeks and 8 weeks, the gastroenemius muscles of each group were taken for histological test. CD31 was detected by immunohistochemistry, and the microvessel density of each group was counted. Results Significant difference was observed between 4 weeks and 8 weeks in the microvessel density from the three hydrogel groups （P〈0.05）. At 4 weeks, there was no significant difference between the PPP group （ 8.67± 1.66 ） and PBS group （7.33 ± 1.66 ） （P〉0.05）, but the SA-CS group （ 15.33 ±3.64 ） and slanc group （ 13.78 ±2.99 ） was significantly higher than the PBS group（P〈0.05 ）. At 8 weeks, compared with PBS group（8.72±1.36）, the microvessel density of PPP group（14.39±1.33）, SA-CS group（23.33±2.11 ） and slanc group（18.61±1.72） was significantly increased （P〈0.05）. The SA-CS group showed higher levels than slanc group on microvessel density. Conclusion The three synthetic hydrogels have the ability to promote the regeneration of blood vessels, in which sodium alginate and chitosan eomposited hydrogel has more obvious regeneration of blood vessels. It has potential clinical application in tissue engineering repair, organ tissue regeneration, and ischemic disease treatment.