自体脂肪干细胞局部注射治疗复杂性肛瘘的临床与相关实验研究

【作者】 章阳；

【导师】 江滨；

【作者基本信息】 南京中医药大学 ， 中医外科学（专业学位）， 2023， 博士

【摘要】 目的:1、评价ADSC临床治疗复杂性肛瘘的患者的有效性并验证其安全性。2、探索ADSC联合化湿健脾方治疗大耳兔肛瘘模型的治疗作用及其可能的作用机制。方法:1、本前瞻性病例对照研究选取2018年1月至2019年12月在南京中医药大学附属南京中医药医院肛肠科招募的24例复杂性肛瘘患者。患者根据需要分为ADSC组和ERAF组。其中ADSC组接受自体ADSCs局部注射治疗,ERAF组接受经肛门直肠黏膜瓣推移术治疗。治疗后第12周经临床评估和和磁共振或经直肠超声检查确认瘘管愈合情况;治疗后3-12个月随访评估复发情况。记录治疗前后患者的视觉模拟评分(VAS评分)和肛门失禁评分(Wexner评分)并监测治疗全程出现的所有不良事件用于评估治疗的安全性。2、选用年龄6-12月,体重2-4kg的大耳兔共20只,采用丝线联合铁质导丝穿透括约肌法构建经括约肌瘘模型,6周造模成功后将大耳兔随机分为4组,每组各5只。Sham组:无任何操作、Model组:造模后予以无菌盐水局部注射和冲洗、Model+ADSCs组:造模后接受脂肪干细胞治疗,即予以局部(内口区域和管壁)注射,每个瘘管分别注射2×106个ADSCs、Model+ADSCs+JPHS组:造模后接受ADSCs治疗的同时,在造模成功开始时起即予化湿健脾方200ml/d灌胃,直至治疗结束。使用HE染色法观察治疗后第7、14、30、60d各组组织病理学变化情况;PKH26荧光标记法示踪各组中ADSCs含量;免疫组织化学法检测各组创面组织中α-SMA蛋白的表达情况;ELISA法用于检测各组创面肉芽组织中 VEGF 的表达量;α-SMA、COL1、TGF-β1、THBS1、VEGF mRNA 的表达则使用 qRT-PCR进行评估。结果:1、第12周ADSC组和ERAF组的闭合率分别为54.5%(6/11)和53.8%(7/13),两组间差异无统计学意义。ADSC组术后第5天VAS评分显著低于ERAF组[1(0,2)VS2(2,4),p=0.011],其他时间差异无统计学意义。24例患者的Wexner评分无明显变化,两组间差异无统计学意义。ADSC组不良事件发生率(27.27%)低于ERAF组(53.85%),但两者差异无统计学意义(p>0.05)。ADSC组复发率(18.2%)高于ERAF组(7.7%),但差异不显著(P=0.439>0.05)。2、HE染色结果显示:治疗后与Sham组相比,Model组的瘘管未愈合,肉芽组织增生严重,脂肪组织被破坏,并被大量炎症细胞浸润。Model+ADSCs组的瘘管组织中可见许多新生组织,肉芽组织减少,脂肪组织增加,并出现了新的毛囊腺,但是仍然有炎症细胞浸润和充血。Model+ADSCs+JPHS组的瘘管肛门组织中可见丰富的脂肪组织和毛囊腺,肉芽和炎症细胞基本不见。荧光染色结果显示:观察第14d,Model+ADSCs+JPHS组中PKH26的荧光显著高于Model+ADSCs组。与Sham组相比,Model组中α-SMA表达量明显减少(P<0.001)。与 Model 组相比,Model+ADSCs 及 Model+ADSCs+JPHS 组中 α-SMA表达量显著增加(P<0.001),并且Model+ADSCs+JPHS组中α-SMA表达量明显高于Model+ADSCs组(P=0.0069)。第3d时,Model组创面肉芽组织VEGF含量明显低于Shαm组(P=0.001),第 3、7、14d 中 Model+ADSCs 组和 Model+ADSCs+JPHS 组的 VEGF 含量明显高于 Model 组(P<0.001)。创面组织中,Model+ADSCs+JPHS组α-SMA、COL1、TGF-β1、VEGF mRNA表达量高于model组(P<0.01),而THBS1 mRNA表达量则低于model组,且更显著(P<0.001)。结论:(1)ADSC是治疗复杂性肛瘘一种安全、有效的方法,其疗效优于ERAF,并且具备保护患者肛门功能,疼痛轻,并发症少,恢复快,患者耐受性好等多种优势。(2)ADSC联合健脾化湿方治疗可能是未来临床治疗肛瘘的可行方法,健脾化湿方能够增强ADSC促进生长因子分泌、改善血管生成、促进胶原纤维形成的作用从而加速肛瘘术后的创面愈合。更多还原

【Abstract】 Objective:1.To evaluate the clinical safety and efficacy of ADSC in the treatment of complex fistula-in-ano.2.Explore the therapeutic effect and possible mechanism of ADSC combined with Hua Shi Jian Pi Decoction in the treatment of fistula-in-ano with a model of Japanese rabbits.Methods:1.Totally,24 patients with complex CPAF were enrolled in this prospective case-control study from January 2018 to December 2019 in the National Colorectal Disease Center of Nanjing Hospital of Chinese Medicine Affiliated to Nanjing University of Chinese Medicine.Patients were divided into ADSC group and endorectal advancement flap(ERAF)group according to their desire.The healing of fistulas(healing of all treated fistulas at baseline,confirmed by doctor’s clinical assessment and magnetic resonance imaging or transrectal ultrasonography)was evaluated at week 12 after treatment.And assessment of recurrence at follow-up of 3-12 months after treatment.In addition to their safety evaluation based on adverse events monitored at each follow-up,the patients were also asked to complete some scoring scales at each follow-up including pain score with visual analog score(VAS)and anal incontinence score with Wexner score.2.A total of 20 Japanese rabbits aged 6-12 months and weighting 2-4kg were selected to construct a transsphincter fistula model using silk thread combined with iron guide wire penetration method.After successful modeling at 6 weeks,the Japanese rabbits were randomly divided into 4 groups,with 5 rabbits in each group.Sham group:no operation,Model group:only local injection and flushing of sterile saline after modeling,Model+ADSCs group:only treatment with adipose stem cells after modeling,i.e.local injection(in the oral area and tube wall),2 injections per fistula×106 ADSCs,Model+ADSCs+JPHS groups:After modeling,while receiving ADSCs treatment,from the successful start of modeling,Hua Shi Jian Pi Decoction 200ml/d was administered by gavage until the end of treatment.HE staining method was used to observe the postoperative histopathological changes in each group from the 7th,14th,30th,and 60th day after treatment;PKH26 fluorescence labeling method was used to trace the content of ADSCs in each group;Immunohistochemical detection of wound tissue in each group α-The expression of SMA protein;ELISA method was used to detect the expression level of VEGF in granulation tissue of wounds in each group;QRT PCR detection α-SMA,COL1,TGF-β1.The expression levels of THBS1 and VEGF mRNA.Results:The closure rates within ADSC group and ERAF group at week 12 were 54.55%(6/11)and 53.85%(7/13),respectively,without significant difference between them.VAS score in ADSC group was significantly lower than that in ERAF group at the 5th day postoperatively[1(0,2)VS 2(2,4),p=0.011],but no differences were observed at the other time.Wexner score of all patients was not increased with no significant differences between the two groups.Adverse events were observed fewer in ADSC group(27.27%)than that in ERAF group(53.85%),but there was no significant difference between them(p<0.05).The recurrence rate in the ADSC group(18.2%)was higher than that in the ERAF group(7.7%),but there was no significant difference between them(P=0.439).2.HE staining results showed that after treatment,compared with the Sham group,the fistula in the Model group did not heal,with severe proliferation of granulation tissue,destruction of adipose tissue,and infiltration of a large number of inflammatory cells.In the fistula tissue of the Model+ADSCs group,many new tissues were observed,including a decrease in granulation tissue,an increase in adipose tissue,and the appearance of new hair follicle glands.However,there were still inflammatory cell infiltration and congestion.The fistula anal tissue in the Model+ADSCs+JPHS group showed abundant adipose tissue and follicular glands,while granulation and inflammatory cells were almost absent.The fluorescence staining results showed that on the 14th day of observation,the fluorescence of PKH26 in the Model+ADSCs+JPHS group was significantly higher than that in the Model+ADSCs group.Compared to the Sham group,the expression level of α-SMA was significantly reduced in the Model group(P<0.001).While in the Model+ADSCs and Model+ADSCs+JPHS groups,the expression level of α-SMA significantly increased compared to the Model group(P<0.001),and the expression level ofα-SMA in the Model+ADSCs+JPHS group was significantly higher than that of the Model+ADSCs group(P=0.0069).On the 3rd day,the VEGF content in the wound granulation tissue of the Model group was significantly lower than that of the Sham group(P=0.001).On the 3rd,7th,and 14th day,the VEGF content in the Model+ADSCs group and Model+ADSCs+JPHS group was significantly higher than that of the Model group(P<0.001).The mRNA expression level of α-SMA,COL1,TGF-β1,VEGF in the wound tissue of M odel+ADSCs+JPHS group was higher than that of the model group(P<0.01),while the mRNA expression level of THBS1 was significantly lower than that of the model group(P<0.001).Conclusions:(1)ADSC is a feasible and effective treatment for complex fistula-in-ano,which is not inferior to that of ERAF.It has multiple advantages such as protecting anal function of patients,relieving pain,reducing complications,allowing quick recovery,and being well-tolerated.(2)The combination of ADSC and Jian Pi Hua Shi Decoction may be a feasible method for future clinical treatment of anal fistula.Jian Pi Hua Shi Decoction can enhance the role of ADSC in promoting growth factor secretion,improving angiogenesis,and promoting collagen fiber formation,thereby improving wound healing after anal fistula surgery.更多还原