背景:细胞凋亡在肠缺血再灌注损伤和修复中起主要作用,参附注射液对肠缺血再灌注时肠上皮细胞凋亡的影响有待观察.目的:分析肠上皮细胞凋亡与肠缺血再灌注损伤和修复特征的关系.设计:随机对照动物实验.单位:湖北省咸宁市中心医院普外科及武汉大学人民医院普外科.材料:实验于2005-03/08在咸宁市中心医院中心实验室完成,选择54只健康雄性大鼠,体质量200～250 g,购自武汉大学医学院动物实验中心.方法:随机摸球法将大鼠分为对照组6只,肠缺血再灌注组24只及参附处理组24只.①麻醉大鼠,分离其肠系膜上动脉,肠缺血再灌注组、参附处理组用无损伤血管夹阻断肠系膜上动脉血流1 h,再灌注1,24,72 h,参附处理组于阻断前30 min经静脉输注参附注射液8 mL/(kg·h),20mL/(kg·d),由人参和黑付子组成(雅安三九药液公司,批号:030302).对照组不阻断肠系膜上动脉血流,对照组与肠缺血再灌注组于阻断前30 min经静脉输注等量生理盐水,整个实验过程对大鼠进行面罩给氧.②肠缺血再灌注组和参附处理组分别于缺血后1 h、再灌注1,24,72 h取材,对照组于造模后取材,每组各6只.观察各组大鼠肠黏膜组织病理学改变(采用下列评分系统:0分,绒毛和腺体正常;1分部分绒毛顶端上皮轻度受损;2分,上皮下腺体轻度受损;3分,上皮下间隙扩大,毛细血管充血;4分,上皮与固有层中度分离,腺体受损;5分,部分绒毛顶端脱落;6分,绒毛明显脱落;7分,固有层绒毛脱落;8分,固有层开始消化分解;9分,出血、溃疡形成)和有丝分裂活性.③采用末端脱氧核苷酸转移酶介导的dUTP缺口未端标记实验结合激光共聚焦显微镜观察小肠黏膜上皮细胞凋亡情况,测定细胞凋亡指数,即每张切片随机取10个高位视野,每100个细胞中凋亡阳性细胞数.④在苏木精-伊红染色的肠黏膜上皮细胞间观察有丝分裂相,10个相邻肠黏膜上皮细胞间具有有丝分裂相的细胞数作为有丝分裂活性指数.主要观察指标:各组大鼠组肠黏膜组织病理学改变,细胞凋亡指数和有丝分裂活性的测定.结果:纳入大鼠54只全部进入结果分析.①参附处理组大鼠肠缺血1 h、再灌注1,24 h黏膜组织病理学改变评分分别为[(0.65±0.35),(3.87±0.86),(0.65±0.35)分,低于肠缺血-再灌注组[(7.11±1.01),(8.05±1.34),(1.53±0.48)分,P＜0.05].②参附处理组大鼠肠缺血1 h、再灌注1,24,72 h黏膜上皮细胞凋亡指数分别为(17.24±7.05),(24.20±9.87),(11.49±4.71),(6.02±2.16)个,低于肠缺血-再灌注组[(51.09±13.76),(54.89±15.58),(23.54±9.64),(12.47±5.52)个,P＜0.05],缺血1 h、再灌注1 h有丝分裂活性分别为(10.37±2.03),(11.72±2.07)个,高于肠缺血-再灌注组[(8.24±1.69),(9.95±1.93)个,P＜0.05].结论:参附注射液能够降低肠黏膜上皮细胞凋亡,并增强肠黏膜上皮细胞有丝分裂活性,从而促进肠黏膜损伤的修复.
揹景:細胞凋亡在腸缺血再灌註損傷和脩複中起主要作用,參附註射液對腸缺血再灌註時腸上皮細胞凋亡的影響有待觀察.目的:分析腸上皮細胞凋亡與腸缺血再灌註損傷和脩複特徵的關繫.設計:隨機對照動物實驗.單位:湖北省鹹寧市中心醫院普外科及武漢大學人民醫院普外科.材料:實驗于2005-03/08在鹹寧市中心醫院中心實驗室完成,選擇54隻健康雄性大鼠,體質量200～250 g,購自武漢大學醫學院動物實驗中心.方法:隨機摸毬法將大鼠分為對照組6隻,腸缺血再灌註組24隻及參附處理組24隻.①痳醉大鼠,分離其腸繫膜上動脈,腸缺血再灌註組、參附處理組用無損傷血管夾阻斷腸繫膜上動脈血流1 h,再灌註1,24,72 h,參附處理組于阻斷前30 min經靜脈輸註參附註射液8 mL/(kg·h),20mL/(kg·d),由人參和黑付子組成(雅安三九藥液公司,批號:030302).對照組不阻斷腸繫膜上動脈血流,對照組與腸缺血再灌註組于阻斷前30 min經靜脈輸註等量生理鹽水,整箇實驗過程對大鼠進行麵罩給氧.②腸缺血再灌註組和參附處理組分彆于缺血後1 h、再灌註1,24,72 h取材,對照組于造模後取材,每組各6隻.觀察各組大鼠腸黏膜組織病理學改變(採用下列評分繫統:0分,絨毛和腺體正常;1分部分絨毛頂耑上皮輕度受損;2分,上皮下腺體輕度受損;3分,上皮下間隙擴大,毛細血管充血;4分,上皮與固有層中度分離,腺體受損;5分,部分絨毛頂耑脫落;6分,絨毛明顯脫落;7分,固有層絨毛脫落;8分,固有層開始消化分解;9分,齣血、潰瘍形成)和有絲分裂活性.③採用末耑脫氧覈苷痠轉移酶介導的dUTP缺口未耑標記實驗結閤激光共聚焦顯微鏡觀察小腸黏膜上皮細胞凋亡情況,測定細胞凋亡指數,即每張切片隨機取10箇高位視野,每100箇細胞中凋亡暘性細胞數.④在囌木精-伊紅染色的腸黏膜上皮細胞間觀察有絲分裂相,10箇相鄰腸黏膜上皮細胞間具有有絲分裂相的細胞數作為有絲分裂活性指數.主要觀察指標:各組大鼠組腸黏膜組織病理學改變,細胞凋亡指數和有絲分裂活性的測定.結果:納入大鼠54隻全部進入結果分析.①參附處理組大鼠腸缺血1 h、再灌註1,24 h黏膜組織病理學改變評分分彆為[(0.65±0.35),(3.87±0.86),(0.65±0.35)分,低于腸缺血-再灌註組[(7.11±1.01),(8.05±1.34),(1.53±0.48)分,P＜0.05].②參附處理組大鼠腸缺血1 h、再灌註1,24,72 h黏膜上皮細胞凋亡指數分彆為(17.24±7.05),(24.20±9.87),(11.49±4.71),(6.02±2.16)箇,低于腸缺血-再灌註組[(51.09±13.76),(54.89±15.58),(23.54±9.64),(12.47±5.52)箇,P＜0.05],缺血1 h、再灌註1 h有絲分裂活性分彆為(10.37±2.03),(11.72±2.07)箇,高于腸缺血-再灌註組[(8.24±1.69),(9.95±1.93)箇,P＜0.05].結論:參附註射液能夠降低腸黏膜上皮細胞凋亡,併增彊腸黏膜上皮細胞有絲分裂活性,從而促進腸黏膜損傷的脩複.
배경:세포조망재장결혈재관주손상화수복중기주요작용,삼부주사액대장결혈재관주시장상피세포조망적영향유대관찰.목적:분석장상피세포조망여장결혈재관주손상화수복특정적관계.설계:수궤대조동물실험.단위:호북성함저시중심의원보외과급무한대학인민의원보외과.재료:실험우2005-03/08재함저시중심의원중심실험실완성,선택54지건강웅성대서,체질량200～250 g,구자무한대학의학원동물실험중심.방법:수궤모구법장대서분위대조조6지,장결혈재관주조24지급삼부처리조24지.①마취대서,분리기장계막상동맥,장결혈재관주조、삼부처리조용무손상혈관협조단장계막상동맥혈류1 h,재관주1,24,72 h,삼부처리조우조단전30 min경정맥수주삼부주사액8 mL/(kg·h),20mL/(kg·d),유인삼화흑부자조성(아안삼구약액공사,비호:030302).대조조불조단장계막상동맥혈류,대조조여장결혈재관주조우조단전30 min경정맥수주등량생리염수,정개실험과정대대서진행면조급양.②장결혈재관주조화삼부처리조분별우결혈후1 h、재관주1,24,72 h취재,대조조우조모후취재,매조각6지.관찰각조대서장점막조직병이학개변(채용하렬평분계통:0분,융모화선체정상;1분부분융모정단상피경도수손;2분,상피하선체경도수손;3분,상피하간극확대,모세혈관충혈;4분,상피여고유층중도분리,선체수손;5분,부분융모정단탈락;6분,융모명현탈락;7분,고유층융모탈락;8분,고유층개시소화분해;9분,출혈、궤양형성)화유사분렬활성.③채용말단탈양핵감산전이매개도적dUTP결구미단표기실험결합격광공취초현미경관찰소장점막상피세포조망정황,측정세포조망지수,즉매장절편수궤취10개고위시야,매100개세포중조망양성세포수.④재소목정-이홍염색적장점막상피세포간관찰유사분렬상,10개상린장점막상피세포간구유유사분렬상적세포수작위유사분렬활성지수.주요관찰지표:각조대서조장점막조직병이학개변,세포조망지수화유사분렬활성적측정.결과:납입대서54지전부진입결과분석.①삼부처리조대서장결혈1 h、재관주1,24 h점막조직병이학개변평분분별위[(0.65±0.35),(3.87±0.86),(0.65±0.35)분,저우장결혈-재관주조[(7.11±1.01),(8.05±1.34),(1.53±0.48)분,P＜0.05].②삼부처리조대서장결혈1 h、재관주1,24,72 h점막상피세포조망지수분별위(17.24±7.05),(24.20±9.87),(11.49±4.71),(6.02±2.16)개,저우장결혈-재관주조[(51.09±13.76),(54.89±15.58),(23.54±9.64),(12.47±5.52)개,P＜0.05],결혈1 h、재관주1 h유사분렬활성분별위(10.37±2.03),(11.72±2.07)개,고우장결혈-재관주조[(8.24±1.69),(9.95±1.93)개,P＜0.05].결론:삼부주사액능구강저장점막상피세포조망,병증강장점막상피세포유사분렬활성,종이촉진장점막손상적수복.
BACKGROUND: Apoptosis plays a key role in intestinal mucosal ischemia-reperfusion injury and recovery; meanwhile, effect of shenfu injection on apoptosis of intestinal epithelial cells during intestinal mucosal ischemia-reperfusion injury should be studied further.OBJECTIVE: To investigate the relationship between the apoptosis of intestinal epithelium and characteristics of intestinal mucosal ischemia-reperfusion injury and recovery.DESIGN: Randomized controlled animal experiment.SETTING: Department of General Surgery, Xianning Central Hospital;Department of General Surgery, Renmin Hospital of Wuhan University.MATERIALS: The experiment was carried out in the Central Laboratory,Xianning Central Hospital from March to August 2005. Fifty-four healthy male SD rats weighting 200-250 g were provided by Animal Center of Medical School, Wuhan University.METHODS: The rats were divided randomly into 3 experimental groups:control group (n=6), ischemia-reperfusion group (n=24) and shenfu treatment group (n=24). ① Pentobarbital sodium solution (40 mg/kg) was administrated into the intraperitoneal cavity to induce anaesthesia. Through a midline abdominal incision, the mesenteric blood vessel of a 15-cm segment of mid-intestine was occluded for 60-minute with an atraumatic vascular forceps. The control group underwent the same procedure except for unblocking the mesenteric blood vessel. At the end of 60 minutes ischemia period the forceps was removed to allow reperfusion, the abdominal cavity was closed. ShenFu injection (8 mL/kg ·h, 20 mL/kg ·d, produced from Yaan Three-Nine Pharmaceuticals Co, No: 030302) was injected 30 minutes before occlusion in SF treatment group, same quantity of 0.9% natrii chloride was injected in control group and ischemia-regeneration group at the same time, and oxygen was inbreathed during the operation and ischemia-regeneration. ② Experimental intestinal canals were sampled for the following analysis when all groups were respectively performed sham ischemia for 1 hour, intestinal ischemia for 1 hour and reperfusion for 1, 24and 72 hours. Sections were observed in light microscope. Histological mucosal damage in each sample was evaluated as followed scoring system: 0score, normal muscosal villi and gland; 1 score, slight lesion near the tip of the villi; 2 scores, slight lesion of subepithelial gland; 3 scores, development of subepithelial (Gruenhagen) spaces near the tip of the villi with capillary congestion; 4 scores, extension of the subepithelial space with moderate epithelial lifting from the lamina propria; 5 scores, a few denuded villous tips; 6 scores, massive denuded villi; 7 scores, denuded villi with exposed lamina propria and obvious gland lesion; 8 scores, disintegrateon of the lamina propria; 9 scores, haemorrhage and ulceration. ③ The Tunel method (TdT mediated biotin-dUTP nick and labeling; TdT-Frag EL DNA fragmentation detection kit) was used. Inbrief, this method allowed the identification of apoptosis nuclei in tissue samples through DNA fragment and labeling. Apoptosis Index (AI) was set as the average number of apoptosis cells in per 100 cells by observing ten high power fields of adjacent villi and crypts. ④ The mitotic phase of crypt epithelial nucleus within intestinal mucosa was observed in intestinal sections stained with haematoxylin and eosin. The number of cells with nucleus mitotic phase was counted in ten adjacent mucosal crypts, which was taken as the index of mitotic activity of intestinal mucosal epithelial cell.MAIN OUTCOME MEASURES: Intestinal mucosal histopathological changes, apoptosis of intestinal mucosal epithelial cell and mitotic activity of intestinal mucosal crypt.RESULTS: All 54 rats were involved in the final analysis. ①) Scores of histopathological changes were (0.65 ±0.35) points in 1-hour ischemia group, (3.87±0.86) points in 1-hour reperfusion group and (0.65±0.35)points in 24-hour reperfusion group; which were lower than those in ischemia-reperfusion group [(7.11±1.01), (8.05±1.34), (1.53±0.48) points; P＜ 0.05]. ② Indexes of apoptosis were 17.24±7.05 in 1-hour ischemia group, 24.20±9.87 in 1-hour reperfusion group, 11.49±4.71 in 24-hour reperfusion group and 6.02±2.16 in 72-hour reperfusion; which were lower than those in ischemia-reperfusion group (51.09±13.76, 54.89±15.58,23.54±9.64, 12.47±5.52; P ＜ 0.05). Activities of mitosis were 10.37±2.03and 11.72±2.07 in 1-hour ischemia group and 1-hour reperfusion group,respectively; which was higher than those in ischemia-reperfusion group(8.24±1.69, 9.95±1.93; P ＜ 0.05).CONCLUSION: Shenfu injection can significantly attenuate apoptosis of intestinal epithelium, increase crypt mitotic activity, and promote intestinal epithelium regeneration or repair.
