从脂肪抽出物的脂类和液体部分提取脂肪来源干细胞的细胞生物学研究

目的 探索从抽吸物中脂质和液体部分分离、体外培养脂肪组织来源干细胞的新方法,并通过其生长动力学、形态学、分化能力、细胞衰老和表面标记物轮廓5个方面的特征进行鉴定比较.方法 抽脂术后抽吸物分解为脂质和液体部分.脂质和液体部分分别用酶消化法和直接离心过滤法分离、培养ASCs,观察其在体外培养的形态学和生物学特点;MTT比色法测细胞活性,统计学分析;流式细胞仪测定细胞周期;随机选取3、4、6、8代做丫啶橙染色检测细胞的衰老;用流式细胞仪、免疫组织化学染色法鉴定其表面分子表达;成脂、成骨定向诱导分化,油红O染色、茜素红染色定性.结果 从吸脂抽吸物的脂质和液体两部分中都能培养出大量的ASCs,分别为PLA和LAF,呈成纤维细胞样贴壁生长,MTY测定细胞活性及细胞周期研究发现PLA、LAF这两种细胞的活力与增殖能力是非常相似的;丫啶橙染色3、4、6、8代细胞无明显衰老;流式细胞仪检测显示干细胞标志的CD29、CIM4、CD34的表达均呈阳性;免疫化学染色发现Ⅷ因子、CD31、CD105、SMA表达阳性;成脂诱导分化2周后,细胞内可见有大量脂滴,油红O染色可见胞浆内有大量红染颗粒.成骨诱导2周后,细胞可见白色矿化钙盐沉积,茜素红染色可见成骨细胞红染.结论 本实验建立了一种自人体脂肪抽吸物中脂质和液体部分分离和培养ASCs的新方法,经济简便实用,从成人脂肪抽吸物液体部分中也可以分离得到大量的可为脂肪组织工程所利用的ASCs,其细胞量与脂质来源的ASCs的量基本相同.贴壁的LAF与PLA细胞在细胞的生长动力学、形态学、细胞衰老、表面标志物和分化能力等方面具有非常相似的特性,都具有很强的增殖活性且衰老率较低,能稳定表达干细胞表面标志并能实现定向成脂、成骨多向诱导分化.这种经过最小限度人工干预的ASCs可能是将来脂肪组织工程比较理想的种子细胞之一.     

In vivo osteochondrogenic potential of cultured cells derived from the periosteum

Periosteal cells were isolated from young chicks, introduced into cell culture, subcultured, and then inoculated into athymic, nude mice to test the in vivo osteochondrogenic potential of cultured periosteal cells. In monolayer cultures, the adherent periosteal cells showed fibroblastlike morphology and overtly expressed neither osteogenic nor chondrogenic phenotypes. Cultured cells inoculated heterotopically into nude mice eventually gave rise to bone tissue at the subcutaneous injection site. The process of bone formation occurred through two different mechanisms: intramembranous bone formation at the peripheral portion of the inoculum early and endochondral bone formation in the central portion later. Frozen, preserved periosteal cells also formed bone after introduction into nude mice in the same temporal histologic sequence as the unfrozen cells. Cultured chick muscle fibroblasts from donors that were the same age as controls did not form bone or cartilage when inoculated under identical conditions to those of cultured periosteal cells. These results suggest that periosteum of young chicks contains subsets of progenitor cells that possess the potential to differentiate directly into osteoblasts or chondrocytes when inoculated in vivo. Importantly, this potential is retained after enzymatic isolation, cell culture, subculturing, and freeze preservation.     

The viability of fatty tissues within adipose aspirates after conventional liposuction: a comprehensive study

This study was conducted to evaluate the viability of fatty tissues within adipose aspirates after conventional liposuction and to examine their potential role as a source of donor material for possible future autogenous fat grafting. Samples of adipose aspirates (group I, n = 8) were obtained from adult female patients who underwent a conventional liposuction of the abdomen. Samples of fresh fatty tissues obtained from adult female patients who underwent an abdominoplasty (group II, n = 8) were cut into small pieces and served as a control. All samples were spun at 50 x g for 10 minutes; fatty tissues were then collected from the middle layer after centrifugation for the following studies: trypan blue vital staining for viable fatty cell counts, glycerol-3-phophatase dehydrogenase (G3PDH) assay for functional evaluation of fatty tissues, and routine pathology for histology of fatty tissues. There was no significant difference of viable fatty cell counts in group I compared with group II (2.57 +/- 0.56 versus 2.74 +/- 0.59 x 10/mL, P = 0.56). G3PGH assay showed a marked decrease of the enzyme activity in group I compared with group II (0.34 +/- 0.13 versus 0.76 +/- 0.13 micro/mL, P < 0.0001). Histologically, the normal structure of fatty tissues was found primarily in both groups. Our results indicate that although fatty tissues within adipose aspirates after conventional liposuction maintain normal structure with near the same number of viable fatty cells compared with fresh ones, they have a less-than-optimal level of cellular function and may not survive well after they are transplanted.     

Tangential injuries and hematoma. Application of liposuction combined with biological glue]

The authors report an unconventional method of management in hematomas complicating tangentiel trauma. They use the application of liposuction within the first days, this procedure allow complete removal of the clots. It is associated with a biological glue sprayed all over the skin detachment. This simple operative technique is carried out under local anaesthesia and minimizes the recurrence of hematoma. The long term morphological result is satisfactory, avoiding the sequelae of conventional procedures.     

Effects of cytokines on cultured microvascular endothelial cells derived from gerbil brain

Microvascular endothelial cells were isolated from gerbil brain and cultured. These cells retained an endothelial-specific marker, FVIII-related antigen. Alkaline phosphatase activity was also present in early passage. Two weeks after plating, these cells were attached to the culture dishes and had become like cobblestones in appearance. Then, the addition of tumor necrosis factor at a concentration of 1000 U/ml or more suppressed the DNA synthesis activity of endothelial cells by about 70% and induced morphological changes in the cells, which developed a spindle-like form and showed overlapping of cells, indicating loss of contact inhibition. The administration of interferon-tau induced no change. When a similar experiment was performed using culture supernatants of human glioma cells that had been cultured for a few days, DNA synthesis activity was suppressed by approximately 50% or more in 6 of 12 samples. The suppression of activity, however, was abolished by the addition of anti-tumor necrosis factor antibody in these 6 cases, suggesting the presence of activity resembling that of the tumor necrosis factor in the culture supernatants.     

超量灌注吸脂术在减少并发症方面的研究

为了探索可以减少并发症的脂肪抽吸术，分别运用超量灌注钝性技术及锐性技术对离体脂肪组织块进行抽吸。通过组织切片观察抽吸后皮下残留结构及脂肪抽吸物的差异。认为正确的选用手术方法及手术器械能保留皮下脂肪组织中的血管，神经，纤维隔，从而可以减少或避免并发症的发生。     

人正常主动脉瓣、风湿性主动脉瓣间质细胞的生物学行为比较

目的　比较人正常主动脉瓣、风湿性主动脉瓣瓣膜间质细胞体外培养的生物学特性。方法　从人正常主动脉瓣和风湿性病变主动脉瓣组织分离培养瓣膜间质细胞 ,在细胞形态学和生长动力学方面进行比较研究。结果　体外培养的正常主动脉瓣瓣膜间质细胞与风湿性主动脉瓣瓣膜间质细胞在形态学方面差异无显著性 ,但风湿性病变瓣膜间质细胞排列更加紊乱 ,极性消失 ,有交叉重叠现象 ,细胞浆内有较多残余小体。在接种相同的细胞密度和相同的培养条件下 ,两者细胞生长率为 (2 .0 5± 0 .1 0 ) %、(2 .1 3± 0 .1 2 ) %、分裂指数为 (2 1 .32± 1 .46)‰、(2 4 .2 5± 1 .98)‰、克隆形成率为 (45 .2 0± 2 .50 ) %、(48.60± 2 .82 ) %差异无显著性 (P >0 .0 5)。结论　正常与风湿性病变主动脉瓣瓣膜间质细胞在体外培养状态下 ,其形态学和生长动力学 ,具有相似的生物学特性     

肿胀麻醉下外超声与非超声吸脂术的比较研究

目的：比较肿胀麻醉法下的外超声吸脂与单纯负压吸脂术的差别，以探讨外超声吸脂术的优点，方法：将10－例吸脂者的腹部均分为左右两侧，在肿胀麻醉下一侧行外超声吸脂，另一侧行单纯负压吸脂，比较吸脂效果并分析吸出物，结果：临床吸脂患者中，外超声作用侧所吸出的上层脂肪占总吸出量的百分比大于非超声吸脂侧（P＜0．001），吸出物中外超声吸脂侧血红蛋白浓度低于非超声吸脂侧（P＜0．001），三酰甘油浓度高于非超声吸脂侧（P＜0．001），血红蛋白与三酰甘油的比值低于非超声侧（P＜0．001），提示外超声吸脂明显较非超声吸脂术脂肪乳化好，出血亦少，且操作省力，术后并发症轻，皮肤收缩效果好，结论：外超声吸脂术是一种去除局部堆积脂肪的更为安全，有效的方法。     

外周血来源间充质干细胞/内皮祖细胞复合细胞膜片的构建及其生物学特性初步研究

目的从外周血中分离获取外周血来源间充质干细胞(peripheral blood mesenchymal stem cells,PBMSC)和外周血内皮祖细胞(peripheral blood endothelial progenitor cells,PBEPC),构建复合细胞膜片并初步研究其生物学特性。方法抽取健康成年新西兰大白兔外周血,采用密度梯度离心法分离获取PBMSC和PBEPC,分别行形态学观察及鉴定。取第3代PBMSC和PBEPC以1∶1比例进行成膜诱导形成复合细胞膜片,取单纯第3代PBMSC同法制备单一细胞膜片。取两种细胞膜片行HE染色,观察细胞膜片内细胞分布情况;分别对两种细胞膜片行成骨诱导,收集诱导1、5、10 d的上清液,利用ELISA法检测两种细胞膜片上清液内ALP、骨钙素(osteocalcin,OCN)和VEGF的表达情况。结果PBMSC细胞形态单一,呈纺锤形或多角形,具备良好的成骨、成脂分化能力;PBEPC细胞形态单一,呈铺路石样,成管试验阳性。两种细胞膜片均呈白色半透明膜状物。HE染色示,与单一细胞膜片组相比,复合细胞膜片组膜片更厚,具备更多细胞层数及更高的细胞密度。ELISA法检测示,随诱导时间延长,两组细胞膜片上清液中ALP、OCN和VEGF表达量均有所增加。复合细胞膜片组诱导培养5、10 d OCN表达量显著高于单一细胞膜片组,10 d ALP表达量显著高于单一细胞膜片组,1、5、10 d VEGF表达量均显著高于单一细胞膜片组,差异均有统计学意义(P<0.05);其余各时间点两组间各蛋白表达量比较差异均无统计学意义(P>0.05)。结论PBMSC具备稳定的MSCs分化能力,因其取材微创具备良好的应用前景。通过与PBEPC共培养、成膜诱导等手段构建复合细胞膜片,为组织缺损的修复提供了一种新的思路与探索。     

Neointimas cultured in vitro for circulatory assist devices: I-Comparison of cultured cells derived from autologous tissues of various organs

In order to develop an antithrombogenic surface for cardiovascular prostheses, neointimas were produced from various autologous tissues on velour-lined and flat prosthetic surfaces. Twenty-four mongrel dogs and six calves were used and three modalities of tissue culture techniques, i.e. explant culture, subculture, and direct trypsinization followed by in vitro culture, were applied. It was found that good neointimas can be made in vitro from autologous skeletal muscle, vein, omentum and granulation tissue on velour-lined prostheses. For the silicone rubber prosthesis with flat surface, the omentum was the only usable tissue to produce a living cell lining in vitro because of easy procurement, short preparation time, formation of uniform and thin cell layers, and least incidence of bacterial contamination.     

正常皮肤,增生性瘢痕和瘢痕疙瘩成纤维细胞培养,生物 …

目的 探讨正常皮肤、增生性瘢痕和瘢痕疙瘩成纤维细胞的体外培养、生物学特征及超微结构、阐明其应用价值。方法 采用成纤维细胞体外培养技术，从正常皮肤成纤维细胞与增生性瘢痕，瘢痕疙瘩成纤维细胞在细胞增殖，细胞形态，细胞遗传学特征及细胞超微结构方面进行比较研究。结果 体外培养的正常皮肤成纤维细胞与增生性瘢痕、瘢痕疙瘩成纤维生长率和形态学相同，细胞遗传学特征和超微结构相似。结论 据此结果和其他学者的观点，认     

Cutaneous retraction. Data from liposuction and other clinical procedures]

The remarkable capacity of the skin to stretch is used in the skin expander technique. In parallel, the skin is also able to retract, which is very useful for the plastic surgeon. This ability to retract may open the road for new techniques. Suction lipectomy, which allows removal of large volumes of fat without skin resection, is a privileged technique for the study of retractability characteristics. This study compares the pre and postoperative conditions in 392 cases of body liposuction and 108 cases of cervicofacial liposuctions, to determine the factors which influence skin retraction. It appears that age and the total amount of fat removed have an incidence on the time needed to obtain the final result. However the treatment site emerges as a determinant factor on the potential of the skin to retract postoperatively. In practice, the skin elasticity appears to be very different in different treatment sites, and considerable variations can even be observed between two neighbouring regions. In the neck and cheeks the skin seems considerably more retractable than in other sites, and we can trace the boundaries of the optimal retractability for the liposuction: "the golden triangle at the neck". The considerable plasticity of the skin overlying the neck contrasts with the poor ability of facial skin to retract. On the body, the skin resumes its previous form in the vast majority of patients (90%). These data, derived from a liposuction study, may be compared with other clinical observations.(ABSTRACT TRUNCATED AT 250 WORDS)     

NBD多肽对小鼠树突状细胞成熟状态的影响

目的观察NBD多肽对脂多糖（LPS）刺激的小鼠骨髓源性树突状细胞（DC）生物学活性的影响。方法培养小鼠骨髓源性DC，分为对照组、LPS组及NBD组，对照组不予任何处理，LPS组加入终质量浓度为1mg／L的LPS，NBD组于加入NBD50txmol／ml，4h后给予1mg／LLPS刺激，继续培养3d。流式细胞术检测DC细胞表面CD80、CD86及MHC-11分子的变化，ELISA法检测各组DC分泌肿瘤坏死因子-α（TNF-α）、干扰素-7（IFN-γ）和白细胞介素．12（IL-12）的浓度，混合淋巴细胞培养检测T细胞增殖能力，免疫细胞化学及Westernblot检测DC核因子-kB（NF-KB）的表达。结果LPS可促进DC高表达CD80、CD86及MHC-11分子，促进Th1型细胞因子释放、NF-KB高表达并易位于核内，并诱导T细胞增殖，NBD多肽可阻断LPS的这些效应。结论NBD多肽可抑制DC成熟，增强同种未成熟DC的免疫耐受诱导作用，为进一步研究DC的临床应用奠定了基础。     

A study of perforin appearing in human lymphokine-activated killer (LAK) cells derived from spleen and peripheral blood mononuclear cells]

Perforin is regarded as one of the main cytotoxic factors in such cells. We succeeded in cloning human perforin cDNA and recently used it to assess perforin appearing in LAK cells. To derive the LAK cells, mononuclear cells separated from a human spleen were mixed with recombinant interleukin-2 and cultured. Almost no perforin mRNA appeared on day 0 but definitely accelerated on day 1 and tended to decrease on and after day 2. Peak of perforin mRNA observed on day 1 was found to precede cytotoxic activity on K562 and Daudi cells by one day. Similar results were found in LAK cells derived from peripheral blood mononuclear cells. Regarding the expression of perforin mRNA on day 0 as 1, the values on day 1 and 2 were calculated as 3.6 and 1.8, respectively. Immunological staining using an antiperforin antibody revealed perforin in the cytoplasm of large cells formed blasts, this also confirmed derivation of perforin on the protein level. Flow cytometry indicated that cells containing perforin included most CD16+ NK cells and some CD8+ T lymphocytes. The fact that TNF and IFN were simultaneously derived together with perforin suggested that the collective joint effect of these substances results in cytotoxic activity.     

Comparison of prostanoid synthesis in cultured human vascular endothelial cells derived from omentum and umbilical vein

Endothelial seeding of vascular grafts may reduce thrombogenicity and significantly improve graft patency. For clinical studies endothelial cells derived from human omentum may be the ideal source of cells as they are obtainable in large numbers. This study was designed to assess their ability to produce the antithrombogenic substance prostacyclin (PGI2). Human omental microvascular endothelial cells (HOTMECs) were grown and their ability to produce prostacyclin (PGI2), PGE2, PGF2 alpha and thromboxane A2 (TXA2) in response to a range of agonists was measured by radioimmunoassay. Control experiments were performed using human umbilical vein endothelial cells (HUVECs). Both HUVECs and HOTMECs synthesised similar quantities of PGI2 basally and both increased production after adding A23187 (calcium ionophore), arachidonic acid, sodium fluoride and phorbol ester. In contrast, both thrombin and histamine were potent stimulators of PGI2 release in HUVECs but were without effect on HOTMECs. In both cell types serotonin, carbachol and noradrenaline were without effect. The pattern of release of PGE2, PGF2 alpha and TXA2 were identical to those of PGI2 in both cell types but the quantities released were lower. These results show that HOTMECs can produce the antithrombogenic agent PGI2 in significant quantities and that they do so by mechanisms similar to those of large vessel endothelium. This supports the proposal that they would be a suitable cell source for vascular graft seeding.     

大鼠骨髓间充质干细胞的分离培养和生物学特性研究

目的 探讨体外分离培养大鼠骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)的方法,并研究其生物学特性.方法 取大鼠股骨和胫骨,以Ficoll密度梯度离心法结合贴壁法分离纯化大鼠BMSCs,传代扩增,倒置显微镜进行细胞形态学观察,MTT法测定细胞的生长曲线,流式细胞仪细胞表面抗原.结果 原代分离的BMSCs,培养48 h开始贴壁,胞体由圆形变为椭圆形、多角形或短梭型;培养第12天,见胞体渐变为长梭型,并达90%单层融合;经传代扩增,细胞进一步纯化.7代以前,细胞在2 d内处于潜伏期,第3天进入对数生长期,第7天进入平台期;10代后增殖速度变慢;流式细胞仪鉴定BMSCs表而抗原,CD44、CD90、CD34阳性率分别为99.62%、95.13%、2.06%.结论 Ficoll密度梯度离心法结合贴壁法有效分离纯化大鼠BMSCs,且细胞生长稳定,增值能力活跃,具有MSCs的一般生物学特性.     

Fas Mcab对病理性瘢痕成纤维细胞生物学活力的影响

目的　探讨FasMcab对病理性瘢痕成纤维细胞生物学活力的影响。方法　以体外培养的病理瘢痕成纤维细胞为研究对象 ,应用MTT法 ,3H -TDR核苷掺入法 ,观察了FasMcab对增生性瘢痕成纤维细胞活力的影响 ;应用流式细胞仪检测FasMcab作用下 ,增生性瘢痕和瘢痕疙瘩两类成纤维细胞的凋亡率。结果　①低浓度FasMcab对增生性瘢痕成纤维细胞的活力具有促进作用 ;②FasMcab在 4 μg/ml的浓度下可迅速诱导增生性瘢痕成纤维细胞的凋亡 ,但在相同条件下对瘢痕疙瘩成纤维细胞不具有诱导凋亡的作用。结论　瘢痕疙瘩成纤维细胞对FasMcab诱导的凋亡存在抗性。深入研究抗性产生的原因将有助于揭示病理性瘢痕增生的形成机理。