大鼠胰岛的分离纯化方法改进与功能鉴定

目的 通过改进胰腺消化和分离的技术条件,提高成年大鼠胰岛分离纯化产率和质量. 方法 用胶原酶Ⅺ液灌注消化成年SD大鼠胰腺,对胰岛分离纯化方法加以改进:以 4 种比重的 Euro- Ficoll (F1∶D=1.132,F2∶D=1.108,F4∶D=1.069) 和 Hank's 液(F5∶D=1.023) 不连续密度梯度离心,以离心半径 15 cm,2 000 r/min 于4℃缓慢升降离心 20 min,收集位于F1 和 F2界面的胰岛.双硫腙特异染色法鉴定胰岛纯度;二醋酸酯荧光素/碘化丙啶染色法计算胰岛成活率;放射免疫分析法检测葡萄糖刺激的胰岛素分泌量,计算刺激指数.将胰岛当量(islets equivalent quantity,IEQ) 为 1000 的胰岛移植于同品系糖尿病大鼠肾包膜下,9d 内隔日观察动物血糖的变化,评价胰岛功能.比较分离条件优化前后收获胰岛的产率和质量. 结果 改进纯化方法后每只大鼠胰岛收获量为(920±122) IEQ,胰岛纯度> 90%,胰岛细胞成活率为 91%±2%.胰岛细胞功能良好,在低糖和高糖刺激后培养液中胰岛素浓度分别为(18.25±0.32) mU/L 和(36.70±3.57)mU/L,刺激指数为 2.01±0.15.1000 IEQ 胰岛移植于糖尿病大鼠肾包膜下,观察期内可维持动物血糖水平正常. 结论 改进后的胶原酶灌注消化和不连续梯度离心方法提高了胰岛的产率,保证了胰岛的高纯度及高成活率.     

胰岛分离、纯化制备的改进

目的 探讨大型哺乳动物胰岛机械化大量分离、纯化的方法,为人类胰岛移植物的大量制备摸索创造条件.方法 应用改进的机械化胰岛分离、纯化系统,用HCA和UW液顺序原位灌洗犬胰腺,主副胰管插管,4℃胶原酶-V(1.5 g/L)+胰酶抑制剂pefabloc(0.4 mmol/L)灌注后,Ricordi-Chamber消化罐消化,4℃COBE2991连续密度梯度离心纯化,测定胰岛当量(IEQ)、胰岛纯度及存活率、胰岛素及C-肽的释放量、培养24 h后光镜及电镜观察.结果 胰腺消化时间为(25.0±6.0)min,胰岛外分泌腺包裹率为(9.4±2.4)%,消化后胰岛收获量为(17.2±3.6)×104IEQ/每个胰腺,纯化后胰岛收获量为(8.3±2.0)×104IEQ/每个胰腺,胰岛纯度为(89.7±3.5)%.纯化胰岛体外低糖与高糖刺激下胰岛索分泌量及C-肽的释放量良好,培养24 h后形态结构及功能正常.结论 本实验室改进的胰岛机械分离方法及各设备运行可靠,获得的胰岛形态功能良好,可望用于临床人类胰岛的大量制备.     

小鼠胰岛的分离纯化及体外功能检测的实验研究

目的：探讨获得高质量小鼠胰岛的分离纯化方法，评价其功能。方法：采用胆总管内胶原酶灌注膨胀消化胰腺的方法分离小鼠胰岛，不连续密度梯度离心法纯化胰岛，用双硫腙（Dithizone，DTZ）对胰岛进行特异性染色计算胰岛产量及纯度，以葡萄糖和茶碱刺激胰岛素释放检测胰岛功能。结果：胰岛的产量和活性主要与胰腺均匀膨胀和胶原酶的消化时间有关。平均每个小鼠胰腺能得到150～250个高质量胰岛，活性〉95％，纯度〉90％。葡萄糖及茶碱（carbachol，Cch）刺激后胰岛素释放量明显增加。结论：改良的胆总管内胶原酶灌注膨胀消化小鼠胰腺及不连续密度梯度Ficoll-400纯化胰岛的方法，可获得产量较高、纯度及功能较好的胰岛。     

大鼠胰岛分离纯化技术的改良及活性研究

目的胰岛的纯度和活性对胰岛移植治疗糖尿病的疗效有很大影响。探讨一种大鼠胰岛分离纯化的新方法,以获得高纯度、高产量、活性好的胰岛。方法选取健康成年雄性SD大鼠10只,体重250～300g,逆行胆总管灌注Ⅴ型胶原酶溶液,38℃水浴消化约15min后,采用两种方法纯化胰岛细胞:A组采用Ficoll400不连续密度梯度液,B组采用Ficoll-PaqueTMPLUS溶液。行双硫腙(dithizone,DTZ)染色鉴定胰岛并计算胰岛当量(islet equivalent quantity,IEQ)、胰岛纯度,锥虫蓝染色检测胰岛活性。取B组胰岛用海藻酸钠-聚左赖氨酸-海藻酸钠(alginate/poly-L-lysine/alginate,APA)包裹制备胰岛微囊,体外静止葡萄糖刺激胰岛素释放实验检测微囊化和未微囊化胰岛的生物学活性。结果DTZ染色示胰岛呈猩红色,有圆形、椭圆形和不规则形,胰岛边缘清晰,大部分直径为50～300μm。A、B组IEQ值分别为338.04±76.61和834.80±54.00,比较差异有统计学意义(P0.05)。A、B组胰岛纯度分别为88.31%±2.67%和95.63%±1.96%,差异无统计学意义(P0.05)。A、B组胰岛活率分别为67.40%±5.15%和86.05%±2.52%,差异有统计学意义(P0.05)。胰岛APA微囊囊形呈完整圆形,大小均匀,微囊直径为1.5～2.0mm,每个微囊中包裹1～3个胰岛。葡萄糖刺激释放胰岛素实验显示,未微囊化胰岛和微囊化胰岛在低糖下的胰岛素分泌浓度分别为(5.53±1.64)ng/mL和(4.76±0.26)ng/mL,高糖下分别为(11.95±2.07)ng/mL和(14.34±3.18)ng/mL,高糖刺激下胰岛素释放量为低糖刺激的2倍多,差异有统计学意义(P0.05);两组的刺激指数分别为2.16±0.30和3.01±0.59,差异无统计学意义(P0.05)。结论Ficoll-PaqueTMPLUS溶液作为纯化液分离纯化胰岛的方法具有操作简便、胰岛产量高、纯度高等优点,获得的胰岛经微囊化或未微囊化体外培养均有良好活性。     

海藻酸钠-氯化钡微囊对大鼠胰岛体外胰岛素分泌功能的影响

目的　观察海藻酸钠 -氯化钡微囊对大鼠胰岛体外胰岛素分泌功能有无影响。方法　以海藻酸钠和氯化钡为材料 ,采用气体吹喷制囊法将新鲜分离纯化的大鼠胰岛制成微囊化胰岛 ,取空微囊、微囊化大鼠胰岛与未微囊化大鼠胰岛各 5 0 0只 ,分为 10份 ,置于培养板中培养 ,用放免法测定并比较第 2、4、6 d培养液中基础胰岛素浓度。结果　空微囊组第 2、4、6 d的基础胰岛素平均浓度均为 0 nm ol/ 1/ 5 0 ,微囊化大鼠胰岛组第 2、4、6 d的基础胰岛素平均浓度为 5 .179、5 .80 6、5 .5 5 8nm ol/ 1/5 0只 ,未微囊化大鼠胰岛组第 2、4、6 d的基础胰岛素平均浓度为 5 .4 4 1、6 .0 80、5 .4 6 8nmol/ 1/ 5 0只 ,后两者差异无显著性意义 (P>0 .0 5 )。结论　海藻酸钠 -氯化钡微囊对大鼠胰岛体外胰岛素分泌功能无影响     

微囊化大鼠胰岛异种移植治疗小鼠实验性糖尿病的研究

目的 研究海藻酸钠－聚赖氨酸－海藻酸钠包裹胰岛进行移植的效果。方法 将Ｗｉｓｔａｒ大鼠的胰腺先行胶原酶胰管内注射消化,然后分离,纯化,所得胰岛经培养后制成微囊包膜的胰岛,微囊直径为０．４￣０．５ｍｍ,每个微囊内包１个胰岛。     

微囊化大鼠胰岛异种移植的实验研究

目的　通过动物实验明确微囊化胰岛是否具有免疫隔离作用。方法　SD大鼠胰腺原位消化 ,Ficoll间断密度梯度离心法纯化、分离胰岛 ,气流吹喷制作海藻酸钠 /聚赖氨酸 /海藻酸钠(APA)微囊化大鼠胰岛 ,比较微囊化与未微囊化胰岛的胰岛素释放试验 ;将微囊化 (实验组 )与未微囊化 (对照组 )大鼠胰岛植入链脲佐菌素 (STZ)诱导的I型糖尿病小鼠中 ,作两组间血糖正常持续时间比较。结果　实验组与对照组的胰岛素释放试验差异无显著性 (P >0 .0 5 ) ;实验组血糖正常持续时间为 2 3～ 6 5d(平均 48d) ,对照组为 3～ 6d(平均 5d) ,两组差异有极显著性 (P <0 .0 1)。已排斥的实验组小鼠腹腔灌洗发现部分微囊化胰岛存活 ,部分已坏死 ,但微囊膜皆完整 ,囊壁无纤维化。结论　微囊具有良好的免疫隔离作用 ,可使胰岛移植物存活时间明显延长。同时推测微囊内移植物死亡与细胞因子、自由基作用或营养不足等有关。     

一种经济高效的SD大鼠胰岛细胞分离技术

目的 建立一种经济高效的大鼠胰岛细胞分离纯化方法,为胰腺的修复重建奠定实验基础.方法 成年雄性SD大鼠25只,体重230～380g,共进行5次实验,每5只大鼠一组进行消化和分离.采用医用复方氯化钠注射液(compound sodium chloride injection, CSCI)经胰总管灌注大鼠胰腺,0.5mg/mL V型胶原酶消化后,分别采用浓度为27.0%、23.0%、20.5%和11.0%的Ficoll 400形成不连续密度梯度介质,离心纯化胰岛细胞.双硫腙(dithizon, DTZ)染色行纯化前后胰岛细胞计数和纯度检测;荧光染料碘化丙啶(propidium iodide, PI)和二乙酸荧光素(fluorescein diacetate, FDA)储存液双染色鉴定胰岛细胞活性;RPMIl640培养基培养3d后,分别用浓度为2.8mmol/L的低糖和25.0mmol/L的高糖行葡萄糖刺激胰岛素释放实验检测胰岛细胞功能.结果 5次实验胰岛细胞消化时间为(13.8±1.6)min.DTZ染色鉴定纯化前胰岛细胞数为(5626±422)个,纯化后为(2914±485)个,纯化后的胰岛细胞数较纯化前明显减少(P<0.01),回收率51.6%±6.0%,每个胰腺收获胰岛细胞数为(583±97)个/只.5次分离获得的胰岛细胞纯度为90.2%±3.4%,活性为81.6%±7.0%.培养3d后,葡萄糖刺激胰岛素释放实验显示:低糖环境下胰岛素水平为(39.7±7.5)EU/L,高糖环境为(116.1±17.4)EU/L,比较差异有统计学意义(P<0.01)刺激指数为3.0±0.4.结论 采用CSCI作为大鼠胰岛细胞分离纯化的主要液体试剂,并采用低浓度V型胶原酶消化,不仅可降低实验成本,同时可获得高质量的胰岛细胞.     

乌司他丁对犬胰岛的保护作用

目的判定在机械分离纯化犬胰岛过程中,胰腺原位灌洗时应用乌司他丁(UTI)的保护作用,观察胰岛的获取产量。方法将20只犬随机分为两组,每组10只。HC-A组:胰腺经腹主动脉原位灌洗时用冷HC-A液2500ml;HC-A UTI组:胰腺原位灌洗时,HC-A液中加用UTI10000U/kg。两组均经主胰管用4℃胶原酶V控压灌注,在RicordiChamber系统中消化,用Ficoll连续梯度离心纯化。记录消化时间、胰岛外分泌腺包裹率、胰岛纯度、纯化后胰岛在体外用低糖与高糖刺激下胰岛素分泌量、C-肽的释放量及收获的胰岛当量(IEQ),并对纯化后的胰岛进行光镜及电镜观察。结果HC-A UTI组与HC-A组在胰腺消化时间、胰岛外分泌腺包裹率、胰岛纯度、纯化后的胰岛在体外经低糖与高糖刺激下胰岛素分泌量、C-肽的释放量以及胰岛的形态和结构上比较,差异均无统计学意义(P>0.05)。HC-A组收获胰岛[(3.42±1.47)×104]IEQ,HC-A UTI组收获胰岛[(6.17±2.86)×104]IEQ,两组差异有统计学意义(P<0.05)。结论在犬胰岛分离和纯化过程中,胰腺获取原位灌洗液中加用UTI能保护胰腺组织,增加胰岛产量。     

大鼠胰岛移植物制备与异种移植

增加胰岛收获量，提高胰岛纯度一直是胰岛移植中面临的重要问题，本实验经胰管注射胶原酶，胰静止消化分离成年大鼠胰岛纯度一直是胰岛葡聚糖离心纯化。纯化后胰岛收获量为６１０－８２０个／胰纯度达９２％；胰岛形态结构完整，内分泌细胞超微结构保持良好，对葡萄糖刺激反应胰岛素释放量是基本分泌水平的８倍；异种移植可逆转实验性糖尿病小鼠的高血糖达一周。     

胸腰椎骨折椎弓根内固定断裂与植骨融合方式的相关性分析

[目的]探讨胸腰椎骨折椎弓根螺钉内固定系统内固定术后,椎弓根螺钉断裂与植骨融合方式之间的关系,以探讨胸腰椎骨折植骨融合的最佳方式。[方法]回顾性研究1995年5月~2005年12月本院脊柱外科收治的胸腰椎骨折病人197例,其中A组单纯内固定(不植骨)患者14例,B组“H”形椎板植骨21例,C组横突间植骨67例,D组椎间、椎内联合横突间植骨95例。[结果]术后随访6~32个月,内固定断裂12例,其中A组4例,B组3例,C组5例,D组0例,4组中D组内固定断裂率显著低于其他3组(P<0.05)。[结论]椎间、椎体内联合横突间植骨重建脊柱三柱的稳定性,符合人体生物力学原理,能有效降低内固定断裂的发生。     

On the measurement of the functional properties of the CFTR

A number of methods are currently employed to assess the functional properties of CFTR channels and their response to pharmacological potentiators, correction of the defective CFTR trafficking, and vectorial introduction of new proteins. Here we review the most common methods used to assess CFTR channel function. The suitability of each technique to various experimental conditions is discussed.     

Preservation of the pylorus and resection of the head of the pancreas

The historical evolution of the pylorus-preservation resection of the head of the pancreas is traced from the first resections early in this century to relative standardization of the operation, to a lowering of the operative mortality, and to an interest in improving nutritional status after resection. There are many theoretical advantages for the function of the upper gastrointestinal tract after pylorus and gastric preservation, such as maintenance of gastric capacitance and equilibration of osmotic pressure in gastric digestants, foodstuff digestion and absorption, and bowel motility. After the pylorus-preserving resection, gastric emptying is normal, pyloric function to prevent duodenal reflux is often normal, and gastric acids and serum levels of duodenal hormones are at normal levels, whereas after standard pancreatoduodenectomy, all of these are often abnormal. No prospective blinded studies have been published comparing nutritional values after the two operative procedures, but evidence is presented of a satisfactory result with regard to gastric capacitance, body weight gain, and lack of postgastrectomy symptoms. An undoubted advantage of the pylorus-preserving feature is a simplification of the operation. These gains are achieved without increase in operative mortality, without increase in the incidence of jejunal ulcer, and without theoretical or actual decrease in value of the procedure as a cancer operation, except in patients with duodenal carcinoma proximal to the ampulla of Vater.     

下颌全牙弓后移量及最后界的测量

目的：研究下颌牙弓的有效后移量及找寻下颌牙弓移动的后界。方法：选取涉及拔除下颌第三磨牙或下颌第三磨牙缺失的病例18例（男6例,女12例）。采用种植支抗牵引下牙弓向远中,治疗完成时所有病例均明确到达下颌牙弓后界,即下颌第二磨牙远中到达下颌升支前缘软组织交界处。应用治疗前后的曲断片测量下颌第二磨牙远中到升支前缘的距离。结果：下颌第二磨牙后移量为（3.49±1.21）mm;治疗后磨牙后间隙的长度为（4.43±0.97）mm。结论：下颌牙弓可确定性地实现整体后移;最大后移量由磨牙后间隙的长度决定;其最后界止于下颌第二磨牙远中与下颌升支前缘软组织交界处。     

The Measurement of the Inclination Angle of the Hamate and Analysis of the Inclination Angle for the Rotation Deformity of the Little Finger in the Fixation of the Carpometacarpal Joint

ObjectiveComplex base fractures of the fifth metacarpal bone and dislocation of the fifth carpometacarpal joint are more prone to internal rotation deformity of the little finger sequence after fixation with a transarticular plate. In the past, we have neglected that there is actually a certain angle of external rotation in the hamate surface of transarticular fixation. This study measured the inclination angle of the hamate surface relative to the fifth metacarpal surface for clinical reference.MethodsIn a prospective single‐center study, we investigated the tilt angle of 60 normal hamates. The study included thin‐layer computed tomography (CT) data from 60 patients from the orthopaedic clinic and inpatient unit from January 2017 to March 2020, including 34 men and 26 women who were 15~59 years old, average 35 years old. The CT data of 60 cases in Dicom format of the hand was input into Mimics and 3‐Matics software for three‐dimensional (3D) reconstruction and measuring the angle α between hamate surface and the fifth metacarpal surface. According to the possible placement of the transarticular plate on the fifth metacarpal surface, we measured the angle β between the hamate surface 1 and the fifth metacarpal surface and the angle γ between the hamate surface 2 and the fifth metacarpal surface.ResultsThe average angle between the hamate surface and the fifth metacarpal surface was 11.66°. The hamate surfaces 1 and 2 have an external rotation angle of 7.30° and 7.51° on average with respect to the fifth metacarpal surface, respectively. There is no statistically significant difference in the angles between the two groups (P > 0.05).ConclusionsThe horizontal angle of the dorsal side of the hamate is different from the back of the fifth metacarpal surface, and the hamate has a certain external rotation angle with respect to the fifth metacarpal surface. No matter how the transarticular plate is placed, the plate always has a certain external rotation angle relative to the fifth metacarpal surface. When the fixation is across the fifth carpometacarpal joint, if the plate does not twist and shape, it will inevitably cause internal rotation of the fifth metacarpal, resulting in internal rotation deformity of the little finger sequence.     

静脉输注甘露醇开放血脑屏障对抗生素透过血脑屏障的影响

目的　通过快速静脉输注甘露醇可逆性开放血脑屏障 (BBB) ,探知此方法能否增加抗生素透过BBB的量 ,在何时达到最高峰 ,其通透量增加后临床上有无不良反应。方法　采用自身配伍设计 ,共 6个样本组。对照组仅使用抗生素 ;其余 5组分别在使用甘露醇前 60、3 0min ,同时使用甘露醇后 3 0、60min使用抗生素 ,各组皆取使用抗生素后 1h的脑脊液测其抗生素浓度。抗生素选用头孢三嗪。结果　测量值经过q检验 ,经 2 0 %甘露醇处理前后的CSF中的头孢三嗪浓度差异有非常显著性。全组患者经临床观察未出现神经系统的不良反应。结论　经静脉快速输注2 0 %甘露醇后可以使透过BBB的水溶性抗生素的量增加 ,两者使用的顺序是在抗生素使用 3 0min内即给予甘露醇快速滴注。该方法不会增加低神经毒性抗生素在中枢神经系统的不良反应。     

Preservation of the pylorus in resection of the head of the pancreas

Whipple's pancreatoduodenectomy was the standard operation for diseases of the head of the pancreas for more than 40 years, but the results were vitiated in part by poor gastrointestinal function and malnutrition. Reintroduced in 1978, pylorus-preserving proximal pancreatoduodenectomy (PPPP) has had an increasing impact on pancreatic surgery as its benefits have been recognized: improved nutritional status, decreased incidence of postgastrectomy syndromes, and a technically easier operation. Postoperative mortality rates and 5-year survival rates are comparable with those of the classic Whipple procedure. PPPP is indicated for most patients with chronic pancreatitis of the pancreatic head. It is also appropriate for patients with periampullary cancer and for those with pancreatic cancer arising from the lower part of ‘the head and the uncinate process. More than 650 patients have now undergone PPPP: 31% for chronic pancreatitis and 66% for periampullary and pancreatic cancers. We assess the indications for PPPP, outline the operation, and review the results.