血竭提取物对角质形成细胞增殖的影响

目的:观察血竭提取物对体外培养角质形成细胞增殖的影响,探讨血竭促进创面愈合的机制。方法:将血竭经过氯仿、乙酸乙酯、乙醇回流提取得到三种提取液,进行角质形成细胞的培养,噻唑蓝(MTT)法检测不同血竭提取物在不同浓度以及不同时间点对体外培养角质形成细胞增殖的影响,并绘制最适浓度下细胞生长曲线。利用流式细胞仪分析最适浓度培养条件下角质形成细胞的细胞周期变化。结果:血竭乙酸乙酯提取物在0.0625～0.5mg/ml浓度范围内,促进角质形成细胞增殖,且呈剂量依赖性,在0.5mg/ml浓度值时促进作用最显著,此条件下细胞DNA合成S期较对照组增加25.7%(P<0.01)。结论:血竭乙酸乙酯提取物具有显著促进角质形成细胞增殖作用,提示血竭可能具有促进创面愈合中再上皮化的作用。     

缓激肽对人角质形成细胞增殖凋亡及分化影响的实验研究

目的观察缓激肽(BK)对体外培养的人角质形成细胞(HKC)增殖、凋亡和分化的影响并探讨其机制。方法以1×10-4~1×10-9mol/LBK作用于体外培养的HKC,采用噻唑蓝法、台盼蓝染色法观察到1×10-4mol/LBK抑制作用最强,以此浓度BK进行余下实验。当HKC达对数生长期后,一部分加入1×10-4mol/LBK作为实验组,另一部分不加BK作为对照组,分别培养24、48h后采用流式细胞仪检测细胞早期凋亡情况及细胞周期,用链霉亲和素-生物素复合物(SABC)免疫细胞化学法检测HKC分化标志物角蛋白10(K10)及内披蛋白的表达情况。用含1×10-4mol/LBK的无血清培养基KC-SFM培养HKC作为实验组,对照组细胞仅加KC-SFM,分别用激光扫描共聚焦显微镜结合钙荧光探针Fluo-3/AM技术检测细胞内游离Ca2 浓度即[Ca2 ]i的变化。结果与对照组比较,实验组G0/G1期细胞比例相对上升34.57%,S期相对下降58.91%,其细胞早期凋亡率为15.34%,明显高于对照组(5.60%,P<0.05)。实验组HKCK10阳性细胞百分比为2.20%,明显低于对照组(6.89%,P<0.05)。BK作用3min后实验组HKC[Ca2 ]i较对照组上升163.0%,之后开始下降,5min后接近对照组。结论高浓度BK可抑制HKC周期进程、明显促进其凋亡及诱导[Ca2 ]i升高,这可能是其使HKC体外生长受抑的部分机制。BK还可抑制表皮再生和HKC分化。     

气-液面培养对角质形成细胞分化的影响

目的 研制角质形成细胞-胶原-猪去细胞真皮基质(PADM)组织工程皮肤,观察气-液面培养对角质形成细胞分化及细胞因子基因表达的影响.方法 将角质形成细胞种植在真皮基质胶原面进行气-液面培养和浸没式培养.采用逆转录-聚合酶链反应(RT-PCR)技术,对比研究两种培养方法获得的细胞膜片bFGF基因表达差异.结果 气-液面培养获得的角质形成细胞层结构与正常皮肤相似,细胞分化良好.单纯浸没式培养角质形成细胞层较薄,分化较差.气一液面培养获得的细胞膜片bFGF mRNA表达量明显高于单纯浸没式培养(t=3.825,P<0.01).结论 气-液面培养可促进角质形成细胞的生长、分化及复层表皮结构形成,影响细胞因子的基因表达.     

培养的正常和瘢痕角质形成细胞上清液对瘢痕成纤维细胞增殖和胶原合成的影响

目的 研究培养的正常角质形成细胞、瘢痕角质形成的细胞的上清液对增生性瘢痕成纤维细胞生物活性和胶原合成的影响。方法 采用MTT、^3H-脯氨酸掺入法、Ⅲ型前胶原放免试剂盒测定不同来源、不同浓茺培养的角度形成细胞上清液，对增生性瘢痕成纤细胞增殖和胶原合成的影响。结果 正常角质形成细胞上清液，可抑制瘢痕成纤维细胞增殖，对细胞内胶原合成有一定的促进作用，但却抑制细胞内胶原向细胞外分泌。瘢痕角质形成细胞上清液，对增生性瘢痕成纤维细胞增殖的抑制作用不明显，甚至还有促进的趋势，对细胞内外胶原合成、分泌皆有明显的促进作用。结论 正常角质形成细胞与瘢痕角质形成细胞分泌的物质对瘢痕成纤维细胞具有不同作用。     

无血清无滋养层培养的人角质形成细胞生物学特征

目的建立一种人角质形成细胞(HKC)无血清、无滋养层培养方法,观察用该方法培养的HKC的生物学特征。方法取5~10岁儿童及20~30岁成人(各5例)环切术后包皮,分为儿童组和成人组。采用二步消化法分离人包皮标本,检测其原代HKC获得数;用KC无血清培养液培养,光镜下观察HKC形态;荧光显微镜下鉴定HKC,并观察其生长速度;用噻唑蓝(MTT)法检测HKC生长曲线,流式细胞仪检测细胞周期。结果儿童组原代HKC获得数为(1.780±0.010)×106/cm2,较成人组(1.490±0.120)×106/cm2高(P<0.01).HKC形态学观察见刚分离的HKC为透亮的小圆形细胞,台盼蓝染色约94%细胞拒染,多次传代的HKC贴壁速度、贴壁率及透亮度明显增加;荧光显微镜下见细胞胞浆呈强黄绿色荧光,细胞核未着色,证明其为KC.儿童组HKC传代次数为(11.0±1.2)次,较成人组(9.2±0.8)次高(P<0.05).HKC生长曲线无明显潜伏期,细胞增殖速度快,扩增倍数高。G1期细胞为36.15%,G2期细胞为25.17%,S期细胞为38.68%,细胞增殖指数为63.85%。结论无血清、无滋养层培养,是一种较理想的培养HKC的方法。     

人角质形成细胞培养的实验研究

目的：探讨实验室条件下人体角质形成细胞的培养技术，为角质形成细胞的多方面研究提供可靠的细胞来源。方法：采用改良的无血清培养技术进行人体角质形成细胞的培养，用显微镜及电子显微镜观察角质形成细胞的形态特征。结果：培养的角质形成细胞在多次传代后仍保持了正常的形态。结论：用改良的培养角质形成细胞的技术可为角质形成细胞进一步的实验和临床提供可靠的、丰富的细胞来源。     

角质形成细胞条件培养液对成纤维细胞增殖与胶原分泌影响的研究

目的 探讨正常的角质形成细胞对成纤维细胞生物学行为的影响。方法 组织块法培养成纤维细胞，分别加入12．5％、25％与50％浓度的角质形成细胞条件培养液为实验组，加入不含血清DMEM为对照组，采用MTT、羟脯氨酸比色法测定成纤维细胞增殖、胶原合成；以流式细胞仪测定50％浓度角质形成细胞条件培养液组及对照组成纤维细胞生长周期。结果细胞增殖测定，各实验组吸光度（A）值与对照组比较，差异均有统计学意义（P〈0．01）；随浓度增高，A值增加，25％及50％浓度组与12．5％浓度组比较，差异有统计学意义（P〈0．01）；25％及50％浓度组间比较，差异无统计学意义（P〉0．01）。胶原合成测定中，各实验组A值与对照组比较，差异无统计学意义（P〉0．01）；各实验组组间比较，差异无统计学意义（P〉0．01）。细胞周期测定见，50％浓度组可明显促进成纤维细胞通过G1／S及S／G1限制点，S期及G1／M期细胞与对照组相比明显增多，G0／G1期细胞与对照组相比明显减少，差异均有统计学意义（P〈0．01）。结论 正常角质形成细胞的上清液可促进成纤维细胞的增殖，但对其胶原分泌影响不明显。     

角质形成细胞和成纤维细胞两步法培养的实验观察

目的应用中性蛋白酶和胰蛋白酶两步消化法原代培养角质形成细胞和成纤维细胞，并进行传代培养。方法取乳鼠背部皮肤，分离出表皮和真皮，加入中性蛋白酶和胰蛋白酶消化，进行原代和传代培养，通过倒置显微镜观察细胞的生长情况。结果原代培养的角质形成细胞和成纤维细胞长成单层且分布均匀；传代培养后排列规律、生长良好。结论两步消化法可以培养出角质形成细胞和成纤维细胞，并且比以往的培养方法更加方便快捷。     

钙浓度变化对体外培养人表皮角质细胞增殖的影响

目的:比较不同钙浓度培养的人表皮角质细胞(HEKs)增殖能力的差异,确定体外培养表皮角质细胞的最适钙浓度.方法:根据培养液中CaCl2浓度不同,将HEKs分为无钙培养组和0.1、0.3、0.5、1.0 mmol/L CaCl2培养组.细胞培养3 h后,加钙离子荧光染料Fluo-3-AM,显微镜观察细胞内的荧光强度,以反映细胞内钙离子浓度的变化;细胞培养2 d后加Hochest33258核染液,荧光显微镜下观察细胞生长形态;培养3 d时镜下观察细胞的生长情况,并分别用流式细胞术和MTT法分析细胞的增殖情况.结果:钙培养可使细胞内钙离子浓度增加,表现为细胞内Fluo-3-AM荧光强度增强,且随着CaCl2浓度的增加,荧光强度亦随之增加;在钙浓度为0.3mmol/L时,细胞的增殖指数最高,为(51.98±14.31)%,增殖活性最强;0.1mmol/L组次之;随着钙浓度的增加,细胞的增殖指数下降,增殖活性受到抑制,0.5～1.0mmol/L钙培养组的增殖指数和增殖活性均明显低于无钙培养组(P均<0.01).结论:不同钙浓度培养影响表皮角质细胞的增殖能力,钙浓度为0.3 mmol/L时细胞的增殖能力最强.     

白芷活性提取物对人角质形成细胞生物学特性的影响

目的体外观察白芷活性提取物对人皮肤角质形成细胞(keratinocytes,KC)生物学特性的影响,初步探讨其促进创面愈合的可能机制。方法取人皮肤KC的HaCaT细胞株,复苏培养取第5代细胞进行实验。取白芷生药95%乙醇提取后,将其活性提取物溶解于含0.25%FBS的DMEM培养液中,稀释至5×10-2、5×10-3、5×10-4、5×10-5g/L,分别与KC培养5 d后采用MTT法测定细胞增殖活性,以含0.25%FBS的DMEM培养作为对照。根据细胞增殖活性确定最佳浓度后,取KC分别采用最佳浓度白芷活性提取液(实验组)及含0.25%FBS的DMEM(对照组)培养。培养48 h后流式细胞仪测定细胞周期,实时荧光定量PCR检测细胞周期相关基因细胞周期素D1(cyclin D1)、凋亡相关基因天冬氨酸半胱氨酸酶3(Caspase-3)mRNA的表达。结果培养5 d后,MTT测定5×10-4、5×10-3、5×10-2g/L浓度可促进KC增殖,吸光度(A)值与对照组比较,差异均有统计学意义(P<0.05);其中5×10-3g/L浓度组A值最高,确定为最佳浓度。实验组S期及G2/M期细胞数量较对照组明显增多,G0/G1期细胞数量明显减少,差异均有统计学意义(P<0.05)。实验组cyclin D1及Caspase-3 mRNA相对表达量均低于对照组,差异均有统计学意义(P<0.05)。结论白芷活性提取物能促进KC增殖,同时下调cyclin D1的表达加快细胞周期进程,下调Caspase-3的表达抑制细胞凋亡,以加快上皮化进程促进创面愈合。     

Prostate cancer imaging with a new monoclonal antibody: A preliminary report

Background: Optimal treatment of prostate cancer depends on accurate staging. Computed tomography (CT) and magnetic resonance imaging have severe limitations, and standard bone scanning can show only destructive osseous metastases. A radiolabeled antibody specific to prostatic adenocarcinoma could theoretically find evidence of soft-tissue metastases and lymph node involvement. Methods: An immunoconjugate (CYT-356) consisting of a murine monoclonal antibody against human prostatic adenocarcinoma bound to a linkerchelator and radiolabeled with indium 111 was administered intravenously to seven patients with documented Stage D adenocarcinoma of the prostate. Planar imaging was done on days 1, 2, and 3 after injection. The CYT-356 scans were compared with standard technetium Tc 99m sulfur colloid bone scans and CT scans. Results: Optimal imaging results were obtained on the 72-h scans. All patients had lesions on both the^99mTc-sulfur colloid bone scan and the CYT-356 scan. The location of the lesions correlated to a great extent. Two patients had positive lesions biopsied, and both biopsies showed the presence of metastatic prostatic carcinoma. There were no side effects from administration of the antibody. Conclusion: In this preliminary study, CYT-356 scanning appears to be a promising agent to accomplish specific staging of prostatic carcinoma.     

Clinicopathological evaluation of renal allograft treated with anti-CD25 monoclonal antibody

Abstract:  Twenty-seven living-donor kidney recipients were treated with the antibody against CD25 as the induction immunosuppressive agent. They did not develop acute rejection within 1 month after transplantation, and mean serum creatinine level at 1 month was 1.0 ± 0.4 mg/dL. There were no findings of acute rejection or drug-induced nephrotoxity in protocol biopsies at 1 month following transplantation. After 1 month had passed, acute rejection occurred in three cases. The pathological grade of acute rejection varied from borderline to grade III by Banff classification. The careful inspection is necessary to find out the occurrences of acute rejection more than 2 months after transplantation because immunological situation has been changing around this period.     

血管内皮生长因子单抗对大鼠肝缺血再灌注损伤的影响

目的研究再灌注期使用血管内皮生长因子单抗(Vascular endothelial growth factor monoclonal antibody,简称VEGFmAb)对大鼠肝缺血再灌注损伤的影响。方法将SD大鼠18只分为两组(每组n=9)。C组:实验对照组,常规行肝缺血再灌注损伤实验;V组:VEGFmAb处理组:在再灌注期使用血管内皮生长因子单抗。于术后2h、24h分别取血测肝功能,术后24h切取缺血肝叶检测热休克蛋白(heat shock protein 70,简称HSP70)RT-PCR表达、肝病理组织学改变,观察生存状态1周。结果与C组比较,V组HSP70的RT-PCR表达未见明显改变;V组ALT、AST在术后明显下降,肝病理组织学改变有所减轻,1周生存率提高(55.56%vs0)。结论再灌注期使用VEGFmAb有助于减轻大鼠对缺血再灌注损伤,改善预后。     

An acrosomal antigen of human spermatozoa and spermatogenic cells characterized with a monoclonal antibody

Monoclonal antibodies were raised against acrosomal antigens of human sperm by immunizing BALB/CA mice with purified ejaculated human spermatozoa. An ELISA-assay, employing glutaraldehyde-fixed spermatozoa as antigen, was used to screen the hybridomas producing anti-human sperm antibodies. Two hybridoma cell-lines produced antibodies which bound to the acrosomal region of spermatozoa. Both gave identical results in preliminary tests and therefore only one was chosen for further experiments. This antibody stained the acrosomal region of fixed but not living spermatozoa by indirect immunofluorescence, indicating an intra-acrosomal localization of the antigen. In acetone-fixed frozen sections of human testis this antigen was expressed only in germ cells in the adluminal compartment of seminiferous tubules. The antigen was clearly visible in round spermatids from the beginning of the cap phase of acrosome development and was also present in premature germ cells which were present in ejaculates and which were in the early stages of acrosome development. By immunochemical analysis this antibody recognized a molecule of 50 K MW as well as other components of 24 to 34 K. The pattern of staining for the antigen was similar in the presence or absence of beta-mercaptoethanol in the sample buffer. The species specificity of the antigen was studied by indirect immunofluorescence using acetone-fixed spermatozoa and the antigen was found to be present in mouse, bovine, ram and boar spermatozoa. This antibody may be useful as an acrosomal marker.     

Antiprostate carcinoma monoclonal antibody (D83.21) cross reacts with a membrane antigen expressed on cytomegalovirus-transformed human fibroblasts

Virological and epidemiological studies have implicated human cytomegalovirus (HCMV) as a possible etiological agent of prostate cancer. Because of the suspected associations, this laboratory tested the reactivity of a prostate-associated monoclonal antibody with HCMV-transformed cells. This mouse monoclonal antibody, D83.21, reacts with a membrane antigen on prostate and bladder tumor cells and does not bind to a variety of other malignant or normal cells. The results of this study indicated that the prostate-associated antibody bound to a membrane antigen on HCMV-transformed cells as detected by radioimmunoassay, immunofluorescence, and complemented-dependent cytotoxicity. This cross-reactivity appeared to be specific for HCMV-transformed cells and did not react with HCMV-infected cells or those transformed by other viruses. Antibody affinity chromatography, used to isolate the D83.21-reactive protein, revealed two peptides of 60 and 28 kd on both prostate tumor and HCMV-transformed cells. The results suggest that D83.21 reacts with a common cell surface protein expressed on HCMV-transformed cells and urogenital tumors.     

Anti-LFA1 monoclonal antibody (25.3) for treatment of steroid-resistant grade III–IV acute graft-versus-host disease

The in vivo efficacy of 25.3 monoclonal antibody (mAb) directed against human LFA1 molecule was assessed in ten patients with steroid-resistant grade III–IV acute graft-versus-host disease (AGVHD). These patients received non-T-cell-depleted allogeneic bone marrow transplantation for aplastic anemia in two cases and hematologic malignancies in eight cases. Five grafts were fully matched, three were one antigen-mismatched, and two were two antigen-mismatched. Despite GVHD prophylaxis with cyclosporin A and short-term methotrexate, AGVHD occurred after a median of 24 days and clearly progressed under prednisone (median 2 mg/kg), given for a median of 12 days. 25.3 mAb was given at a dosage of 0.1 mg/kg in a 4-h perfusion for five daily doses without any clinical or biological side effects. Thirty percent of the patients experienced a reduction in the overall grading with two complete responses. Partial response in at least one involved organ (mostly skin) occurred in 80% of the patients. However, seven out of the eight responding patients experienced a new episode of AGVHD. This observation, which confirms that inhibiting a functional molecule is as efficient as a cytolytic therapy, offers an alternative strategy to antithymocyte globulin (ATG) and cytotoxic mAb in controlling steroid-resistant GVHD.     

抗肿瘤坏死因子-α单克隆抗体减轻骨骼肌缺血再灌注损伤的研究

目的　研究抗肿瘤坏死因子 α(TNF α)单克隆抗体对骨骼肌缺血再灌注损伤的作用。方法　SD大鼠 2 4只 ,建立后肢缺血再灌注模型 ,抗TNF α单克隆抗体用量为 2mg/kg体重。采用酶联免疫吸附试验 (ELISA )法、比色法、免疫组织化学、流式细胞计数及电镜观察分别测定血浆TNF α、组织过氧化物酶 (MPO)、血管内皮细胞粘附分子 (ICAM ) 1、中性粒细胞CD18表达及超微结构改变。结果　应用抗TNF α单克隆抗体后血浆TNF α水平显著降低 (P <0 .0 1)。组织MPO(骨骼肌 :2 .11± 0 .2 5vs 4.2 8± 0 .5 5 ,P <0 .0 1;肺 :0 .93± 0 .0 1vs 2 .62± 0 .10 ,P <0 .0 1)、血管内皮细胞ICAM 1及中性粒细胞CD18(4 8.75± 9.2 8vs 1.13± 0 .2 0 ,P <0 .0 1)均明显下降 ,组织结构损伤减轻。结论　抗TNF α单克隆抗体能减轻骨骼肌缺血再灌注损伤。     

抗角蛋白自身抗体在黑素细胞无血清纯化培养中的应用

目的：探索抗角蛋白自身抗体(AK auto Ab)在黑素细胞纯化培养中的应用，寻找黑素细胞无血清纯化培养的安全而简易的方法。方法：按常规方法制备表皮细胞悬液，应用角质形成细胞无血清培养基(KC-SFM)中加入AK auto hb(用于替代TPA)、bFGF和BPE培养细胞，48h后换液一次，以后则用仅含bFGF和BPE的KC—SFM换液培养。结果：AK auto Ab对角质形成细胞有选择性杀伤作用，用含AK auto hb、bFGF和BPE的KC—SFM进行黑素细胞培养时原代即可获得纯化的黑素细胞。结论：AK auto hb可取代TPA用于黑素细胞无血清纯培养，方法可靠、简单。因AK auto hb是正常人血清中提取的物质，用其培养的黑素细胞在临床移植应用中将更具安全性。     

Anti-CD20 monoclonal antibody treatment of Epstein-Barr virus-induced intrahepatic lymphoproliferative disorder following liver transplantation

Epstein-Barr virus (EBV)-associated post-transplant lymphoproliferative disorders (PTLDs) are a common cause of death in transplant patients. Their incidence following liver transplantation is reported to be between 0.5% and 4%. Despite various therapeutic approaches, there is still no consensus on a treatment strategy. The treatment of transplant recipients with monoclonal antibodies directed against B-cell antigens is a new, therapeutic approach with which, however, little clinical experience has so far been gained. Two patients developed intrahepatic PTLD 7 and 15 months, respectively, after transplantation. In one case, this was diagnosed as polymorphic PTLD, in the other as monomorphic, monoclonal PTLD. After having their immunosuppression terminated, 4 weeks after establishment of the diagnosis, both patients were treated with anti-CD20 antibodies (rituximab) at a dose of 375 mg/m(2) on days 1, 8, 15 and 22. Treatment with rituximab was tolerated well by both patients. One of the patients in whom cholestasis parameters remained high underwent re-transplantation. In one of the cases, the histological work-up confirmed necrosis of 90% of the tumour cells, and complete remission in the other. Both patients died of secondary complications 10 weeks and 10 months, respectively, after the diagnosis of PTLD. We can conclude that treatment of PTLD with Rituximab led to remission in both of our patients. Nevertheless, progression of cholestasis persisted, and both patients ultimately died of complications unrelated to PTLD.