同种瓣的制作与临床应用

目的报告液氮深低温下保存同种带瓣血管的制作方法、组织活性及临床应用效果。方法制作同种瓣24个、抗生素灭菌、梯度降温后置于液氮中保存,并测定冷冻保存后同种瓣的组织活性。同种瓣临床应用5例,其中法洛四联症、肺动脉闭锁2例,先天性主动脉瓣狭窄1例,法洛四联症术后发生室间隔缺损残余漏伴肺动脉瓣重度关闭不全1例,Bentall术后发生感染性心内膜炎1例。结果抗生素灭菌、液氮深低温技术保存同种瓣具有良好的组织活性,糖代谢测定24h葡萄糖消耗大于16mg/dl,组织培养见成纤维细胞生长良好。临床移植5例均成功,术后随访3～8个月,同种瓣无狭窄或关闭不全。结论液氮深低温保存同种瓣安全可靠,临床应用早期效果良好。     

活性带支架绵羊主动脉瓣的制作及组织学和细胞活性研究

目的　制作活性带支架绵羊主动脉瓣 ,观察液氮深低温保存 3个月后的组织结构 ,证实细胞活性 ,为同种二尖瓣置换实验做准备。方法　无菌条件下取 4～ 12个月 ,体重 (4 0～ 60 )kg的绵羊主动脉瓣 8个 ,经修剪、抗菌素灭菌后缝制于弹性支架上、制成绵羊带支架主动脉瓣 ,1℃ /min的速度程控降温至 -80℃后投入液氮冻存。 3个月后解冻 ,取其中 6个 ,剪取瓣叶 ,流式细胞技术评价细胞活性 ,同期冻存的 6个无支架绵羊主动脉瓣对照。另外 2个带支架绵羊主动脉瓣解冻后用光镜及电镜研究其组织结构 ,新鲜主动脉瓣 2个对照。结果　带支架组和无支架组细胞活性分别为 (90 .92± 2 .90 ) %和 (91.69± 3 .5 0 ) %,两组比较差异无显著性 (P >0 .0 5 )。带支架组细胞外基质、内皮细胞及大多数成纤维细胞的超微结构与新鲜瓣膜差异无显著性 ,仅部分成纤维细胞的超微结构有轻微的可逆性改变。结论　带支架设计不影响液氮深低温保存的同种瓣的细胞活性及超微组织结构 ,可用于制作新型的人同种主动脉瓣。     

同种异体腱移植重建膝关节交叉韧带的研究

目的从实验角度观察程序冷冻液氮保存和-80℃深低温保存方法处理同种异体髌腱后重建膝关节交叉韧带的愈合过程并比较其差异。方法将兔的1/2骨-髌腱-骨复合体经-80℃深低温保存和程序冷冻液氮保存2周后观察冻存变化差异并行同种异体移植重建前交叉韧带,分别于术后3周和8周观察细胞毒反应、细胞活性、最大载荷和形态学变化等指标进行比较并与自体移植组对照。结果a）经程序冷冻液氮保存方法处理后,髌腱的最大载荷无明显下降,细胞活性得到了较好的保存,组织学观察冷冻损伤较-80℃深低温保存方法轻微;b）程序冷冻液氮保存处理的移植物在术后未表现明显的排斥反应,且免疫反应随时间的推移而下降;c）移植后3周,各组移植物的最大载荷无显著差异（P〉0.05）,移植后8周,程序冷冻液氮保存组移植物的最大载荷（55.87±1.86）N优于-80℃深低温保存组（52.14±2.79）,而和自体移植组相近（57.70±2.76）N;d）从组织学观察看,-80℃深低温保存组和程序冷冻液氮保存组移植后的愈合过程均和自体移植组相似,而程序冷冻液氮保存组的愈合过程和组织学行为更接近于自体移植组。结论经深低温保存的兔异体髌腱重建膝关节交叉韧带后愈合过程和自体韧带移植重建过程相似。程序冷冻液氮保存法优于传统的-80℃深低温保存法。     

两种方法冷冻保存异体髌腱重建膝关节交叉韧带比较

目的 观察程序冷冻液氮保存和-80℃深低温保存方法处理同种异体髌腱后重建膝关节交叉韧带的愈合过程并比较其差异.方法 将兔的1/2骨-髌腱-骨复合体经程序冷冻液氮保存和-80℃深低温保存2周后,观察冻存变化差异并行同种异体移植重建前交叉韧带,分别于术后3周和8周观察细胞毒反应、细胞活性、最大载荷和形态学变化等指标进行比较并与自体移植组对照. 结果 ①经程序冷冻液氮保存方法处理后,髌腱的最大载荷无明显下降,细胞活性得到了较好的保存,组织学观察冷冻损伤较-80℃深低温保存方法轻微.②程序冷冻液氮保存处理的移植物在术后未表现明显的排斥反应,且免疫反应随时间的推移而下降.③移植后3周,各组移植物的最大载荷差异无显著性,移植后8周,程序冷冻液氮保存组移植物的最大载荷优于-80℃深低温保存组,但和自体移植组相近.④和-80℃深低温保存组相比,程序冷冻液氮保存组的移植物在术后的愈合过程和组织学行为更接近于自体移植组.结论 ①经程序冷冻液氮保存和-80℃深低温保存方法冻存的兔异体髌腱重建膝关节交叉韧带后愈合过程和自体韧带移植重建过程相似.②程序冷冻液氮保存法优于传统的-80℃深低温保存法.     

深低温冷冻保存气管的实验研究

目的　寻找最适于冷冻气管的冷冻保护剂。方法　选择目前常用的几种冷冻保护剂 ,分别冷冻犬气管碎片。经程控降温 ,液氮冻储不同时间后复温 ,通过病理学、电镜以及酶组织化学检查 ,对其活性进行比较。结果　以 10 %二甲基亚砜 0 1mol L海藻糖作为冷冻保护剂冷冻的气管 ,复温后其活性明显高于其它冷冻保护剂 ,且冻储 8个月时气管的活性与冻储 1小时相比仅有微小变化。结论　程控降温 ,液氮冻储可以长期保存气管 ,10 %二甲基亚砜 0 1mol L海藻糖是目前冷冻气管的最佳冷冻保护剂配方。     

同种异体气管材料保存及移植实验研究

目的 探讨同种异体气管材料低温保存方法，效果并经移植验证。方法 20条实验成年犬，麻醉后，不进行气管插管，切取颈段气管6cm，再均分为实验组和对照组，实验组分别进行4℃EC液保存，慢速降温保存（每分钟1℃，5℃，10℃，20℃）和玻璃化降温保存处理，用光镜和电镜进行检查，同时，还对抗冻剂的种类，浓度和降温速率进行了筛选，另用慢速降温保存和玻璃化保存组各10段进行异体气管移植，结果 经10%DMSO（二甲基亚砜）为抗冻剂，降温速度为每分钟1℃和玻璃化法保存的气管样品组织结构完整，并仍具有组织细胞活性，为了证实保存的效果，将经低温保存的气管进行了同种异体移植，其结果较为满意，结论 该方法保存气管制 方法可行，并可应用于临床。     

再次液氮冷冻保存对猪主动脉瓣膜细胞活性及组织结构的影响

目的了解再次冷冻保存对主动脉瓣膜细胞活性及组织结构的影响,探讨液氮冷冻保存的主动脉瓣解冻后再次冷冻保存使用的可行性.方法将猪主动脉瓣叶在抗菌处理后按随机数字表法分成三组,每组6个瓣叶,组Ⅰ作对照,组Ⅱ、组Ⅲ控制降温速率降至-80℃后在液氮中保存,1个月后融化解冻.组Ⅲ解冻并在室温下放置15分钟后更换保存液,再次降温至-80℃后放入液氮中保存,2个月后再融化解冻.采用XTT比色法测定各组瓣膜细胞活性,用免疫荧光组织化学染色、光学显微镜、透射电子显微镜行组织学检测.结果组Ⅱ冷冻保存后瓣膜细胞活性下降到组Ⅰ的63.97%,组织结构一定程度受损;组Ⅲ瓣膜细胞活性下降至组Ⅰ的38.60%,组织结构损害也进一步加重.结论液氮冷冻保存的猪主动脉瓣一经解冻融化,不宜再次冷冻保存使用.     

冷冻预处理温度对胎兔许旺细胞免疫原性的影响

目的 探讨不同维持温度冷冻预处理超深低温保存对胎兔许旺细胞免疫原性的影响.方法 设未冷冻处理细胞为对照组,实验组细胞设计冷冻维持温度为-30℃, -35℃, -40℃, -45℃, -50℃, -55℃, -60℃, -65℃,-70℃,10%二甲基亚砜(DMSO)为低温保护剂,对胎兔许旺细胞进行冷冻预处理,液氮中保存...     

关节软骨细胞库

本文介绍了关节软骨细胞的分离、冷冻保存和复苏技术，并在阐明胶原酶的细胞毒性作用的基础上，提出了分阶段消化法，以减少分离细胞的损伤，由于低温保护剂ＤＭＳＯ也具有细胞毒性，因而应尽量缩短ＤＭＳＯ与软骨细胞在４℃以上的接触时间。降温和复温速度是影响细胞存活的关键因素。降温速度从４℃～－８０℃以Ｉ℃／ｍｉｎ为佳，之后迅速投入－１９６℃的液氮中长期保存，细胞复苏应采用３７℃水浴快速复温法，虽然检测冻存细胞的存活率可采用苔盼蓝拒染试验，但最可靠的方法是细胞培养，利用关节软骨细胞库进行细胞培养、电镜观察和激光流式细胞计量分析，结果证明冻存软骨细胞复苏后仍具有正常的结构和形态并保持新陈代谢和自我复制功能。     

低温保存的嗅鞘细胞移植治疗脊髓损伤的实验研究

目的:观察低温保存复苏后嗅鞘细胞(OECs)局部移植治疗脊髓损伤(SCI)的疗效,探讨低温保存对嗅鞘细胞活性的影响。方法:建立大鼠T10节段脊髓半切损伤模型56只,随机分为四组,每组12只:新鲜OECs移植组(A组),冻存OECs移植组(B组),D/F12培养液移植组(C组)和空白对照组(D组)。A组损失伤局部行新鲜OECs移植,B组将处于对数生长期的OECs冻存3个月,复苏后(用双苯亚甲胺标记)移植到脊髓半切模型大鼠脊髓损伤区。术后第1天、1、2、3、4、6、8及10周时进行联合行为学(CBS)综合评分,术后5周和10周时取材观察移植细胞存活及迁移情况;HE染色及嗜银染色观察脊髓组织病理及轴突情况;NGFRp75免疫组织化学染色情况。结果:术后第1天,2、10周时CBS评分,A组分别为85.78±1.07、58.80±5.00、6.52±2.37;B组分别为86.12±1.29、60.06±6.51、8.15±2.26;C组分别为86.4±1.03、66.28±7.00、30.65±5.60;D组分别为86.75±1.37、69.85±6.61、34.13±5.38。A、B两组间无明显差别(P>0.05);C组与D组间比较无差异(P>0.05);A组及B组较C组和D组在2周后各时段差别有显著意义(P<0.05)。组织学方面,HE染色和嗜银染色A、B两组在术后5周可见多量突起经近侧断端向损伤区域生长,10周时可见神经纤维跨越损伤区域,而C、D组未见有神经纤维跨越损伤区;NGFRp75免疫组化染色,无论5周还是10周,A、B两组在损伤部位均可见阳性染色区域,C、D组未见有阳性着色。术后5周时A、B两组可检测到荧光标记细胞,且可见细胞发生迁移。结论:低温保存的OECs脊髓局部移植治疗SCI与新鲜OECs移植治疗效果无差别。     

Long-term patency of vein grafts preserved in liquid nitrogen in dimethyl sulfoxide.

Autogenous canine jugular veins were stored in 15% dimethyl sulfoxide (DMSO) in liquid nitrogen vapor for one to 28 days and then implanted in the carotid artery as autografts. The patency rate at one year was 62.5-87.5%. The patency rate of fresh jugular vein autografts placed in the carotid artery for one year was 75%. Similar autografts stored in liquid nitrogen vapor for one to 28 days without the cryopreservative DMSO exhibited a zero to 12.5% patency rate at one year. Scanning electron microscope studies revealed preservation of theendothelium in DMSO protected veins and a damaged or sloughed endothelium in veins frozen without DMSO cryopreservation.     

大鼠嗅鞘细胞无血清上清液诱导脊髓神经干细胞分化的研究

[目的]观察大鼠嗅神经鞘细胞上清液对脊髓神经干细胞共培养发生的诱导分化作用。[方法]采用差速贴壁法获得较高纯度的嗅鞘细胞，分时段Mr丌法检测细胞活性，选取最佳状态细胞无血清培养后取上清液，与3代纯化后的神经干细胞共培养，观察分化过程，免疫荧光法鉴定诱导结果。[结果]MTT法分6个时段检测纯化后的嗅鞘细胞，发现9d及12d细胞活性最高。使用无血清嗅鞘细胞上清液与脊髓神经干细胞共培养发现诱导作用明显，向神经元样细胞及胶质细胞分化的比例分别达到53％和42％。[结论]嗅鞘细胞在体外培养的不同时间段活性不同，无血清的嗅鞘细胞上清有明显诱导神经干细胞向成熟神经元分化的作用。     

Investigations of low-temperature storage of articular cartilage for transplantation

Isolated bovine articular cartilage chondrocytes and intact slices of cartilage were investigated to determine the effects of low-temperature cryopreservation on articular cartilage. Studies have focused on prefreezing conditions of cartilage, including the incubation medium and temperature of incubation, type and toxicity of the cryopreservative used, and the penetration of cryopreservative agents into cartilage cells. Cartilage freezing conditions were examined with respect to rate of freezing, controlled differential freezing rates, the ultimate storage temperature, and the time of storage. Cartilage thawing conditions were observed to ascertain the role of membrane osmotic stress during thawing and the effect of variable thawing rates on the viability of chondrocytes. Careful control of these variables can yield cartilage with cellular viability of over 50%. Optimum cryopreservation of viable cartilage should include prefreezing treatment with 7.5%-10% DMSO in nutrient medium, controlled slow freezing to -70 degrees, and rapid thawing in DMSO containing medium. A significant number of chondrocytes in deep-frozen cryopreserved articular cartilage can survive. The work recommends continued clinical use of deep-frozen cartilage.     

Binding regulation of porcine spermatozoa to oviductal vesicles in vitro

In vivo, en route to the site of fertilization, mammalian spermatozoa bind to oviduct epithelial cells (OECs). This binding may favor sperm survival and capacitation, but little is known about the regulation of detachment of sperm from the oviduct in vivo. Therefore, we studied sperm-oviduct interaction in vitro using vesicles formed from OECs in primary culture. Porcine oviducts were collected from gilts at the slaughterhouse. OECs were obtained by compressing the oviduct and culturing them in TCM-199 medium with 10% fetal calf serum for 48 hours. For the first experiment, to test the hypothesis that progesterone (P4) and estradiol (E2) affect sperm-OEC binding, OECs were pretreated for 48 hours with 100 ng/mL of P4 or E2. In the second experiment, porcine follicular fluid (pFF, 5%), caffeine (1 microM), calcium ionophore A23187 (1 microM), and DMSO (0.01%) were added to the incubation medium to provide insights on the mechanisms of sperm release from the oviduct. For both experiments, 50 x 10(6) sperm were coincubated with 50 microL of OEC vesicles in 1 mL of incubation medium for up to 24 hours at 37 degrees C and 5% CO2. After an initial 30 minutes of coincubation, the vesicles settled and a sample of the supernatant was removed to evaluate sperm release and acrosomal status; subsequent samples were removed after 2, 4, and 24 hours of coincubation. To evaluate the effect of the different treatments on sperm integrity, the acrosome status of the spermatozoa was determined using fluoresceinlabeled Pisum sativum agglutinin staining. Experiment 1 showed that P4 pretreatment of OECs interferes with sperm binding compared with pretreatment with E2 or controls (P < .05). In experiment 2, coincubation in the presence of A23187 increased sperm detachment compared with pretreatment with DMSO, pFF, caffeine, or controls (P < .05). For each experiment, the treatments did not affect the percentage of acrosome-reacted sperm compared with controls (P < .05).     

Heating or freezing bone: Effects on angiogenesis induction and growth potential in mice

We have characterized the effect of bone graft treatment by heating or freezing (with or without dimethyl sulfoxide (DMSO)). Tissue culture and dorsal skinfold chambers in mice were used as sites to quantify the effect on angiogenesis, growth and calcification of neonatal femora. Fresh femora increased in both length and cartilage diameter (calcification in vivo only), but cryopreservation or heating abolished the increase in femoral dimensions. in vivo, femora of all experimental groups elicited an angiogenic response from the host tissue, which was most pronounced for fresh femora, weaker for DMSO-pre-served frozen bone and poor for unprotected frozen bone and boiled femora. Freezing in the presence of a cryopreservative (DMSO) was found to preserve the angiogenic potential of frozen bone, whereas unprotected heating or freezing significantly impaired angiogenesis induction and growth potential.     

人胚嗅球嗅鞘细胞及人胚鼻粘膜嗅鞘细胞的分离培养与纯化

[目的]采用差速贴壁法及免疫组化对人胚嗅粘膜OECs及人胚嗅球OECs进行体外纯化培养,探讨建立嗅粘膜OECs及嗅球OECs体外培养的方法.[方法]对差速贴壁后的人胚嗅粘膜OECs及嗅球OECs分别交替应用含13％胎牛血清DMEM - F12培养基进行原代培养.观察嗅鞘细胞的形态学变化,采用p75NTR和GFAP免疫细胞化学染色进行鉴定和纯度检测.[结果]人胚嗅粘膜及人胚嗅球均可培养出嗅鞘细胞,嗅粘膜嗅鞘细胞形态多呈双极、三极,伴有细长的突起.p75NTR和GFAP染色均呈阳性反应,体外培养时人胚嗅球嗅鞘细胞纯度比人胚嗅粘膜嗅鞘细胞高.[结论]差速贴壁法可以分离培养出人胚嗅粘膜嗅鞘细胞及人胚嗅球嗅鞘细胞.     

Cryopreservation of vascularized ovary: an evaluation of histology and function in rats

Cryopreservation of organs has been investigated to sustain the reproductive function of patients undergoing sterilizing chemotherapy and radiotherapy or reproductive surgery. A modified protocol for whole organ cryopreservation was described and the outcome of cryopreservative ovaries was evaluated, and apoptosis of cryopreservative cells stored for different time period and the viability of cryopreserved cells stored at different temperature was examined in rats. Lewis rat ovarian grafts were perfused for 30 min at 0.35 ml/min with M2 medium containing 0.1M fructose and increasing concentrations of 0-1.5M dimethylsulfoxide, cooled to -140 degrees C controlled by a computerized program, and stored in liquid nitrogen (-196 degrees C) for 24 hours. After being thawed, ovaries were transplanted to syngeneic recipients after bilateral oophorectomy. Graft functions were monitored postoperatively. The major findings were that: 1) A 100% survival rate of rat ovaries was achieved in this study. Ovarian hormone secretion recovered in 80% rats which had received cryopreservative ovarian grafts. Postoperative serum estradiol levels in the cryopreservative graft group were lower than in the sham surgery control, but much higher than in the bilateral oophorectomy group. 2) Histological examination of cryopreservative ovarian grafts showed preantral and antral follicles. Two gestations were obtained. 3) Estradiol levels remained low in ovariectomized rats while in the oophorectomized rats given cryopreservative ovarian grafts levels started to rise after 14 +/- 3 days. 4) The average viability in the cells from cryopreservative ovary organ (-196 degrees C) was about 71 +/- 18% compared to 90 +/- 9% of fresh cells. This success should encourage further improvement of cryopreservative techniques for large organs.     

Influence of cryopreserved olfactory ensheathing cells transplantation on axonal regeneration in spinal cord ofadult rats

Objective: To observe the effects of cryopreserved olfactory ensheathing cells (OECs) transplantation on axonal regeneration and functional recovery following spinal cord injury in adult rats. Methods : Twenty-four rats were divided into experimental and control groups, each group having 12 rats. The spinal cord injury was established by transecting the spinal cord at T10 level with microsurgery scissors. OECs were purified from SD rat olfactory bulb and cultured in DMEM ( Dulbecco‘s minimum essential medium) and cryopreserved (-120~C) for two weeks. OECs suspension I (1-1.4) x 105/ul ] was transplanted into transected spinal cord, while the DMEM solution was injected instead in the control group. At 6 and 12 weeks after transplantation, the rats were evaluated with climbing test and MEP ( moter evoked potentials) monitoring. The samples of spinal cord were procured and studied with histological and immunohisto chemical stainings. Results: At 6 weeks after transplantation, all of the rats in both transplanted and control groups were paraplegic, and MEPs could not be recorded. Morphologyof transplanted OECs was normal, and OECs wereinterfused with host well. Axons could regrow into gap tissue between the spinal cords. Both OECs and regrown axons were immunoreactive for MBP. No regrown axons were found in the control group. At 12 weeks after transplantation, 2 rats (2/7) had lower extremities muscle contraction, 2 rats (2/7) had hip and/or knee active movement, and MEP of 5 rats (5/7) could be recorded in the calf in the transplantation group. None of the rats (7/7) in the control group had functional improvement, and none had MEPs recorded. In the transplanted group,histological and immunohistochemical methods showed the number of transplanted OECs reduced and some regrown axons had reached the end of transected spinal cord. However, no regrown axons could be seen except scar formation in the control group. Conclusions: Cryopreserved OECs could integrated with the host and promote regrowing axons across the transected spinal cord ends.     

嗅鞘细胞经低能激光照射后移植对大鼠脊髓损伤神经功能修复的影响

目的 探讨嗅鞘细胞(OECs)经低能激光照射(LPLI)后移植对大鼠脊髓横断损伤后功能修复的影响. 方法 24只SD大鼠T<,12>脊髓完全横断伤模型建模4周后,随机分为OECs移植组、经LPLI的OECs的移植组和空白对照组(DMEM培养液),应用BBB评分法、组织学评价以及Flurogold逆行示踪评价各组脊髓损伤的修复情况. 结果三组大鼠不同移植方式以及评分时间点对移植后下肢功能BBB评分结果的影响差异均有统计学意义(P=0.000),且组间差异均有统计学意义(P=0.000).经LPLI的OECs移植组大鼠头尾侧均可见NGFRp75阳性及GFAP阳性的OECs,OECs移植组则仅可见GFAP阳性的OECs,空白对照组未见NGFRp75阳性及GFAP阳性的OECs.OECs移植组和经LPLI的OECs移植组大鼠头尾侧及瘢痕区均可见Flurogold标记的神经纤维穿行,空白对照组未见Flurogold标记的神经纤维穿行. 结论 OECs及经LPLI的OECs均可促进损伤脊髓的功能恢复,OECs经LPLI后可能更具有促进损伤脊髓功能恢复的作用.     

嗅鞘细胞在周围神经中迁移特性的初步研究

目的 :了解OECs在周围神经中的迁移特性。方法 :从新生乳鼠嗅脑中培养出OECs ,用荧光染色剂hochest标记 ,注入大鼠坐骨神经中 ,在不同的位置对坐骨神经与脊髓造成损伤 ,观察OECs的迁移情况。结果 :1个月后取病理标本观察 ,坐骨神经中损伤一侧OECs迁移距离大于未损伤侧 ,但若损伤在脊髓 ,或脊髓中注射OECs在坐骨神经处进行损伤时对细胞两侧迁移没有影响。结论 :在周围神经中 ,嗅鞘细胞不表现为中枢趋向性 ,而表现为一定的损伤趋向性 ,且其迁移的速度较慢