血管内皮祖细胞促进游离移植脂肪颗粒组织存活的实验

目的探讨血管内皮祖细胞（endothelial progenitor cell，EPC）移植促进游离移植的脂肪颗粒组织的血管新生，提高移植脂肪颗粒组织存活率。方法体外分离、培养人脐血中EPCs，然后与来自人体的脂肪颗粒组织混合移植于裸鼠背部。结果脐血中分离培养的EPCs表达血管内皮细胞生长因子受体（KDR）及细胞表面标记CD34、CD133，EPCs与脂肪颗粒组织混合移植到裸鼠3个月后，EPCs整合到缺血部位新生血管中，与对照组的脂肪颗粒组织存活率分别为（89．3±6．8）％、（42．2±2．5）％（P〈0．05），而且实验组与对照组脂肪颗粒组织周边区毛细血管密度差异有统计学意义（P〈0．05）。术后3个月2组脂肪颗粒组织周边区的EPCs密度分别为（95．2±10．5）个／mm^2、0个／mm^2（P％O．05）。结论体外培养的脐血EPCs移植体内可促进游离移植的脂肪颗粒组织的血管新生，提高存活率。     

脂肪干细胞应用于脂肪移植的新进展

脂肪干细胞(Adipose derived stem cells,ADSC)具有多向分化和自我复制能力,逐渐成为损伤修复及再生医学研究的热点。ADSC能够分化为脂肪细胞以及血管内皮细胞,可分泌细胞因子,促进血管新生,改善炎症损伤的微环境,并抑制细胞凋亡。在脂肪移植中应用脂肪干细胞,可以明显降低移植后脂肪组织的液化吸收,减少不良反应发生率,提高远期疗效。     

辅助脂肪移植技术在促进移植脂肪血管化中的应用进展

自体脂肪组织作为一种理想的软组织填充材料,广泛应用于各种先天疾病及后天创伤所致的颜面畸形矫治、乳房整形以及面部年轻化等手术中,而自体脂肪移植后存在不同程度的脂肪吸收,导致脂肪移植效果不稳定及不可预测,根据移植脂肪细胞的成活机制,早期及时建立有效的血液供应是改善体积保留率的关键。近来在移植前后添加辅助成分促血管新生,进而改善脂肪移植效果成为研究热点,现对辅助脂肪移植技术在促进移植脂肪血管化中的应用进展作一综述。     

ADSCs与PRP联合脂肪颗粒移植对血运重建的影响

目的 探讨兔脂肪来源干细胞(ADSCs)联合富血小板血浆(PRP)对自体脂肪颗粒植入裸鼠皮下后血运重建的影响.方法 体外分离、培养、鉴定兔ADSCs,MTT法检测PRP、全血浆,对照组在不同时段(24、48、72h)对体外培养ADSCs增殖的影响.制备脂肪颗粒、明胶海绵颗粒.体内实验分为五组A组(脂肪颗粒+ADSCs+PRP+明胶海绵);B组(脂肪颗粒+ADSCs+生理盐水+明胶海绵);C组(脂肪颗粒+PRP+生理盐水+明胶海绵);D组(脂肪颗粒+全血浆+生理盐水+明胶海绵);E组(脂肪颗粒+生理盐水+明胶海绵).将各组移植入裸鼠皮下,分别于第7、14、30、90天取材,制作石蜡切片,免疫组化法检测血管数.结果 与其他各组相比,各时间段内A组血管数均明显增多,B、C组相对较多,D、E组血管数最少.结论 ADSCs与PRP联合可促进移植脂肪颗粒中血管生成.     

血管生成素-1对移植颗粒脂肪新生血管的实验研究

血管生成素-1（angiopoietin—tie-1，Ang-1）的作用在于维持血管内皮细胞的成熟及稳定，并在血管生成过程中可促进其出芽及分支，我们旨在探讨，Ang-1是否具有促进移植颗粒脂肪新生血管生成的作用。     

不同剂量雌激素对裸鼠血管瘤模型的影响

目的：建立人毛细血管瘤裸鼠移植模型,探讨血管瘤裸鼠模型建立的最佳条件。方法：将手术切除的1例雌激素受体阳性的儿童增生期血管瘤组织制成组织块,植入20只裸鼠（BALB/cnudemice）皮下,每只4处,将20只裸鼠分为4个实验组。实验1组在移植后给予普通鼠食喂养;实验2组在1组基础上每周肌注雌二醇0.01mg;实验3组在1组基础上每周肌注雌二醇0.1mg;实验4组在1组基础上每周肌注雌二醇1mg,于移植后第30、60、90天切取移植瘤。移植瘤标本进行病理学光镜检查,用血管内皮细胞单克隆抗体CD31、CD34进行免疫组化染色。结果：移植后早期各组标本内皮细胞大量变性、坏死,30天后,单纯喂养的实验1组及实验2组部分移植瘤开始吸收或形成脓肿及纤维化。实验3、4组移植瘤开始缓慢生长。90天后实验1组、实验2组移植瘤均未成活,实验4组移植瘤部分成活,而实验3组移植瘤全部成活。光镜下成活的移植瘤与原血管瘤组织生物学特点相似。结论：不同剂量的雌激素对血管瘤裸鼠移植模型的建立有一定影响,适量的应用雌激素可建立稳定的人血管瘤裸鼠移植动物模型。该模型可以应用到基础和临床的血管瘤研究。     

神经肽Y应用对颗粒脂肪移植存活率的影响

目的：探讨神经肽Y应用对颗粒脂肪移植存活率的影响。方法将成熟颗粒脂肪组织自体移植到兔背部皮下,同时种植含有或不含有神经肽Y的小球,于2周、1个月、3个月后通过大体观察、B超、组织学、免疫组化法分组对照来评价脂肪颗粒的存活情况;同法将人体脂肪颗粒联合神经肽Y异体移植到无胸腺小鼠皮下,观测评价人体脂肪细胞在无胸腺小鼠的存活情况。结果应用神经肽Y组脂肪移植存活率明显高于其他对照组,神经肽Y可以刺激脂肪组织的生长,并且可以增加3个月兔和无胸腺小鼠异体脂肪移植物的存活和血供。结论神经肽Y应用在体内脂肪生成中起了重要作用,可增加脂肪移植存活或者刺激重新脂肪生成,明显提高颗粒脂肪移植的存活率,并可进一步应用于人体,具有良好的临床应用前景。     

基质血管成分与体外扩增的脂肪来源干细胞促进移植脂肪成活的对比研究

目的:比较血管基质成分(st romal vascul ar fract i on,SVF)与体外扩增的脂肪来源干细胞(adi pose-deri ved st emcel l s,ASCs)对移植脂肪成活的促进作用。方法:用手术方法切取家兔腹股沟脂肪,进行ASCs体外分离培养;取第3代ASCs分别进行成脂、成骨诱导实验,CD29和CD31流式鉴定;制备SVF,进行CD29和CD31流式鉴定;脂肪移植裸鼠实验分为3组:SVF、ASCs、和空白对照组(DMEM/F12),每组4只裸鼠,沿背部脊柱两侧对称部位4个移植位点,每组共16个注射移植位点,每点0.3ml脂肪颗粒(adi pose granul e,AG)+0.2ml细胞成分;术后4m时取材称重、固定行HE染色观察移植脂肪组织结构,行CD31免疫组化染色观察新生血管及其密度。结果:家兔ASCs体外分离培养成功,为贴壁生长,第3代细胞形态均呈长梭形。成脂诱导实验油红O染色显示形成脂滴,成骨诱导实验茜素红染色显示形成钙化结节。流式鉴定显示SVF:CD29:17.0%,CD31:1.3%;ASCs:CD29:96.2%,CD31:3.8%。SVF、ASCs和空白对照组各组移植脂肪成活量分别为0.2096±0.0024g,0.1798±0.0033g,0.1350±0.0020g,两两比较差异均有统计学意义,P<0.05。HE染色显示SVF组移植脂肪成活较好,组织结构完整,脂滴大小均一,可见脂肪组织中间有丰富的血管存在;ASCs组移植脂肪成活尚可,组织结构尚完整,脂滴大小一般均一,可见脂肪组织中有结缔组织纤维间隔和新生血管形成;空白对照组结缔组织纤维间隔明显增多,脂滴大小不一,有少量较大空泡形成。各组移植脂肪新生血管密度分别32.6±2.1条/mm2,29.3±1.6条/mm2,23.3±1.9条/mm2,两两比较均有统计学意义,P<0.05。结论:新鲜的干细胞成分SVF比体外扩增的ASCs能更好的促进移植脂肪成活。     

瘦素促进移植颗粒脂肪成活的实验研究

目的　探讨瘦素是否具有促进移植颗粒脂肪组织成活的作用。方法　取大鼠腹部皮下脂肪组织制成颗粒脂肪 ,分别用NS及 2 0ng ml瘦素处理后行自体头皮下移植。在不同观察时间取出移植组织称重 ,同时行HE及血管内皮细胞生长因子 (VEGF)染色。实验结果用SPSS统计分析软件进行统计学分析。结果　实验组和对照组比较 ,用瘦素处理的组织其周围包膜薄 ,脂肪细胞坏死融合少 ,重量维持率在移植 10、2 0、4 0d后分别为 10 8 3%、83 3%、6 6 3% ,和NS处理相应时间组6 0 0 %、4 2 1%、39 5 %的重量维持率相比 ,差异有显著性意义 (P <0 0 5 )。移植组织内VEGF染色强度较高。结论　瘦素对移植鼠颗粒脂肪组织具有促进成活作用 ,其作用机理可能是促进移植组织的血管增生。     

血管基质片段细胞携血管内皮生长因子对移植颗粒脂肪血管化的影响

目的 探讨血管内皮生长因子(vascular endothelial growth factor,VEGF)复合血管基质片段细胞( stromal vascular fraction cells,SVFs)细胞疗法对移植颗粒脂肪组织血管化的影响,寻找一种安全、有效、简单的细胞辅助脂肪移植术式.方法 取患者植皮术后废弃的皮下脂肪,提取SVFs.用DiI染色观察SVFs染色标记情况,锥虫蓝染色观察SVFs活性.将0.3 ml待移植的脂肪颗粒分别与0.2 ml下列细胞混合:①复合有VEGF的SVFs(A组);②SVFs(B组);③DMEM完全培养基(C组).按照随机化原则将3组脂肪组织混合物分别注射移植于裸鼠背部皮下,每只裸鼠背上注射3个点(共12只).观察移植物外形、成活情况、湿重、直径,HE染色及CD31染色镜下观察并计数毛细血管密度.使用SPSS 16.0进行方差分析检验.结果 新鲜提取的SVFs可被DiI标记染色,加用VEGF仍能保持较好的细胞活性,移植术后2个月3组移植脂肪的湿重为:A组(191.90 ±9.81)mg、B组( 177.01±10.50)mg、C组(92.05±8.30)mg,A组＞B组＞C组(P＜0.01).移植物直径为:A组(0.49±0.24)cm、B组(0.40±0.26) cm、C组(0.32±0.28) cm,A组＞B组＞C组(P＜0.01).CD31染色镜下,每高倍镜视野微血管密度:A组(14.58±2.06)个、B组(11.55±2.18)个、C组(7.87±1.55)个,与B组和C组比较,A组较少见纤维结缔组织增生及坏死凋亡细胞.结论 结合VEGF因子的SVFs辅助脂肪移植是一种临床可操作性强、简便可行的较理想的细胞疗法,比单纯SVFs更能有效地促进移植脂肪血管化.     

The effects of estrogen on human breast carcinomas serially transplanted into nude mice

The effect and mechanisms of 17β-estradiol (E₂) on breast cancer cells were studiedin vivo andin vitro, using 5 human breast carcinomas serially transplanted into nude mice. These carcinoma strains consisted of 4 estrogen receptor (ER) positive tumors and 1 ER negative tumor. Mice bearing these tumors were treated with an intramuscular injection of E₂ at a dosage of 50 mg/kg and the tumor doubling time (Td) was calculated in days. The tumor growth was significantly stimulated by E₂ in 3 out of the 4 ER positive tumors, the Td of the E₂ treated groups being 17.6 days for MCF-7 (control: −17.8 days), 12.8 days for R-27 (control: −12.5 days∼14.5 days) and 10.4 days for Br-10 (control: 14.5 days), however, in the T-61 tumor, the growth was inhibited by E₂ in a dose dependent manner. In the case of the ER-negative MX-1 tumor, the tumor cell growth was not affected by E₂. Discrepancies between the effects of E₂ on ER-positive tumors were further analyzed by examining the steroid hormone receptor status and conductingin vitro growth studies.In vitro clonogenic cell assay reproduced the antitumor activity of E₂, indicating that E₂ directly inhibits part of the cell growth of T-61 tumors. The above results suggest that this experimental system provides a useful tool for analyzing the mechanism of estrogen in breast cancer and that the clonogenic assay using ER positive specimens can help to identify breast cancers sensitive to estrogen therapy.     

Changes in the hormone receptors of human breast carcinoma xenografts in nude mice by treatment with cytotoxic agents

We examined the effect of chemotherapeutic agents on the estrogen receptors (ER) of breast carcinomasin vivo using human breast carcinoma strains (Br-10, T-61) serially transplanted into nude mice. When the tumor size reached approximately 1×1×1 cm, mitomycin C (MMC) at doses of 1, 2 and 4.5 mg/kg and cyclophosphamide (CPA) at a dose of 120 mg/kg, were administered once intraperitoneally, and the ERs of the tumors were measured sequentially by the dextran-coated charcoal method. Four days after the MMC administration at above doses, the binding sites of ER in Br-10 were not reduced and binding affinity was not affected. When the changes in ER content with time after the treatment with 4.5 mg/kg MMC and 120 mg/kg CPA were investigated, the ER content was found to be stable until 4 days after the treatment with both drugs, although the growth of T-61 had been significantly inhibited by the drugs. From these findings, it seems reasonable to initiate chemotherapy before endocrine therapy, since the chemotherapeutic agents did not reduce the ER content of the breast cancer strains.     

Growth of human tumor xenografts in nude mice and mice with severe combined immunodeficiency (SCID)

Twenty-three fresh tumor specimens obtained at surgery and 5 serially transferable human tumor xenografts were implanted subcutaneously into nude mice and mice with severe combined immunodeficiency (SCID) to compare the take rates of the fresh surgical specimens and the growth rates of the transferable strains. The overall take rates were 65% for the SCID mice and 60% for the nude mice, without any significant difference, although colon carcinoma seemed to have higher acceptance in the SCID mice with a take rate of 6/8. All the serially transferable strains were successfully accepted in the SCID mice, their growth rates being essentially identical to those in the nude mice. These results indicate that the SCID mouse can be used as a human tumor xenograft-mouse system as well as the nude mouse.     

脂肪注射移植后皮肤质地改善的研究

目的:通过皮下脂肪注射移植,探索移植脂肪对皮肤质地的改善作用。方法:取30只裸鼠,随机分成三组,所有鼠左侧皮下移植人脂肪细胞,在另一侧10只注射硅凝胶,10仅行皮下穿刺,剩下10只无任何处置,8周取皮肤组织做HE、Masson’染色观察皮肤真皮层厚度及真皮层内胶原蛋白的含量。结果:取材时,所有裸鼠上都能看到所移植的脂肪,但体积较术前有较明显缩小。肉眼看,脂肪呈正常外观,有较多新生血管生成。组织学检测,移植脂肪外周有大量细胞外基质形成,实验侧真皮层较对照侧明显增厚,真皮内胶原含量增多。结论:移植的脂肪组织不仅仅是一种填充物,在改善轮廓的同时,还有真皮增厚、胶原含量增加等皮肤改善的效应。     

Human prostatic adenocarcinoma: Comparative experimental treatment of the tumor line pc 82 in nude mice

A human prostatic adenocarcinoma, growing as a transplantable line in nude mice, was subjected comparatively to the usual endocrine treatment schedules including orchiectomy, estrogens, estramustine phosphate, and a study with flutamide (SCH 13521) and cyproterone acetate. The regression under treatment in tumor volume and in the histological pattern is demonstrated, and the possibilities and limitations of the nude mouse model are discussed.     

Sensitivity of anticancer agents on human gastric cancers transplanted into nude mice

In the chemotherapy for gastric cancer, the most sensitive anticancer agent against individual tumors should be prescribed. The establishment of a sensitivity test using nude mice as anin vivo model is urgently awaited by clinicians and researchers alike. Seventy-three tumors derived from human gastric cancer were transplanted subcutaneously into nude mice and these mice were then treated intraperitoneally with anticancer agents. Mitomycin C (MMC), 5-fluorouracil (5-FU) and cyclophosphamide (CPM) were used. The doses given were 3 mg/kg of MMC, 75 mg/kg of 5-FU and 200 mg/kg of CPM. In 52 of the 73 cancers, chemosensitivity was evaluated by the microscopic changes in the tumors. The rate of positive sensitivity against gastric cancer was 44.2% in MMC, 34.6% in 5-FU and 30.8% in CPM, respectively. The sensitivity of each agent tested by this method indicated a good correlation with the clinical therapeutic effects. Our results suggest the feasibility of evaluation of the sensitivity of various agents from the microscopic changes on tumors transplanted into nude mice.     

雌激素受体在椎间盘组织中的表达及意义

【摘要】 目的：通过观察雌激素α、β受体在正常与退变的人椎间盘组织的表达,探讨雌激素受体（ER）与椎间盘退变的关系。方法：根据改良Pfirrmann分级将收集的椎间盘组织分为三组：对照组,外伤导致腰椎爆裂性骨折手术取出的正常髓核组织（Pfirrmann分级1~2级）;观察组,女性腰椎滑脱及腰椎间盘突出症手术取出的退变髓核组织,Pfirrmann分级3～4级为A组,5级为B组。用HE染色法观察对照组和A、B组各15例人腰椎椎间盘髓核组织及髓核细胞的形态学变化;用免疫组织化学染色法（Elivison二步法）检测对照组和A、B组髓核组织中ER-α、ER-β的表达;采用Western-blot法检测对照组和观察组髓核组织中ER-α、ER-β的表达。结果：HE染色示对照组髓核组织中髓核细胞分布均匀,形态完整,未见明显细胞凋亡现象,细胞外基质染色鲜亮,分布均匀;A、B两组髓核细胞分布不均匀,形态不规则,凋亡现象明显,细胞核增大,染色深,细胞外基质减少,色彩灰暗,B组较A组更为明显。免疫组织化学染色示ER-α、ER-β在对照组的髓核组织见有明显棕黄色颗粒,在A、B两组表达减少,以ER-β减少最显著,经统计学分析,A、B两组与对照组间ER-β的表达有显著性差异（P＜0.01）,ER-α的表达较对照组无统计学差异（P＞0.05）,其中A组和B组间无明显差异（P＞0.05）。Western-blot方法检测,ER-α表达A组（0.876±0.058）、B组（0.757±0.045）较对照组（0.885±0.036）降低,但无统计学差异（P>0.05）;ER-β表达A组（0.947±0.043）、B组（0.626±0.042）较对照组（1.275±0.150）显著降低（P＜0.01）,B组较A组表达明显减少（P＜0.01）。结论：在人的髓核组织中的髓核细胞（类软骨细胞）的胞核和胞浆中均存在ER。ER-α、ER-β在退变的椎间盘组织都明显减少,以ER-β最为明显,提示雌激素可能通过ER-β的介导对髓核细胞功能起调控作用。     

Serum CEA levels in patients with gastric carcinoma correlate with the tumorigenicity of their xenografts in nude mice

We examined the correlation among preoperative serum carcinoembryonic antigen (CEA) levels, staining properties of the tumors by CEA immunohistochemistry and the tumorigenicity of their xenografts in nude mice, in 28 patients with gastric cancer. Eleven (40 per cent) of them were positive for serum CEA (≧2.5 ng/ml) and seven (25 per cent) of the xenografts were tumorigenic in nude mice. All the tumorigenic cases were positive for serum CEA (p<0.001) and the mean value of the serum CEA level in the patients with tumorigenic neoplasms was 20.8 ng/ml, being significantly higher than that (1.4 ng/ml) in the patients with nontumorigenic neoplasms (p<0.001). Twenty-five of the 28 carcinomas (89 per cent) were positive for CEA staining in their cancer cells by the ABC method and CEA localization correlated with tumorigencity (p<0.05). These results suggest that the serum CEA level in patients is correlated with the tumorigenicity of their gastric carcinoma xenografts in nude mice and may account for the poor prognosis of patients with high serum CEA.     

Human prostatic adenocarcinoma: Some characteristics of a serially transplantable line in nude mice (PC 82)

Transplantation of tissue from a cribriform human prostatic carcinoma resulted in a serially transplantable tumor line in nude mice, PC 82. During 2 1/2 years the tumor has not changed its histological appearance and remained a moderately differentiated adenocarcinoma. The cells contain large amounts of prostatic acid phosphatase. It has a slow rate of growth and regresses after castration and estrogen treatment. This preliminary information suggests that the PC 82 tumor line may be a suitable model for hormone-dependent human prostatic cancer.