X-盒结合蛋白1条件性基因敲除小鼠模型的建立

目的建立可进行条件性敲除X-盒结合蛋白1(Xbp1)基因的flox杂合子小鼠。 方法通过ET-clone的方法构建基于cre-loxp系统和FLP-frt系统的条件性基因打靶载体。载体线性化后电转JM8A3 ES细胞,经G418和Ganc筛选获得抗性ES细胞克隆。经长片段PCR鉴定获得正确同源重组的阳性克隆。阳性ES细胞经克隆扩增后,注射入C57BL/6J小鼠的囊胚中,获得嵌合鼠,筛选出高比例嵌合小鼠与野生型C57BL/6J小鼠交配后获得阳性F1代小鼠,并分别进行PCR和测序鉴定。 结果成功构建打靶载体,共获得144个抗性ES细胞,克隆经长片段PCR鉴定,共获得4个正确同源重组的阳性克隆。获得4只阳性F1代小鼠,经测序鉴定正确。Xbp1基因flox杂合子小鼠表型无明显异常。 结论成功构建了条件性敲除Xbp1基因的flox杂合子小鼠,为未来研究器官或组织特异性的Xbp1生物学作用奠定了基础。     

生长激素促分泌素受体基因敲除小鼠胚胎干细胞的建立

目的建立生长激素促分泌素受体（ghrelin receptor，GHS-R）基因敲除小鼠胚胎干细胞（ES细胞）杂合子模型，为研究GHS—R基因的功能奠定基础。方法用TK-neo置换原X-pPNT载体的PGK—neo构建目标载体。以小鼠基因组DNA为模板，用PCR方法扩增2条同源臂，将其按照一定方向装入含有TK—neo的X—pPNT载体，并测序鉴定。载体线性化及纯化后电穿孔转染小鼠ES细胞，用G418和更昔洛韦（Gancyclovir）对电穿孔转染后的ES细胞进行正、负筛选培养，得到双药抗性ES细胞，克隆后抽提基因组DNA，分别用PCR方法鉴定2条同源臂，并测序确定成功同源重组的ES细胞克隆。结果改建X—pPNT载体成功，PCR获得2条同源臂片段，测序正确，并按一定方向装入打靶载体，ES细胞转染后经双药筛选得到328个阳性ES细胞克隆，PCR及测序鉴定证实3个克隆发生同源重组。结论本研究成功获得了GHS-R（-／＋）杂合子小鼠ES细胞克隆，为进一步通过显微注射及杂交育种获得GHS—R基因敲除小鼠打下了基础。     

输注分子嵌合前体T细胞减低小鼠脾脏T细胞对异基因小鼠T细胞的刺激反应

目的构建分子嵌合主要组织相容性复合体(MHC)-Ⅰ基因小鼠前体T细胞,并探讨其诱导脾脏T细胞减低对异基因小鼠T细胞反应的可行性。方法体外分离培养BALB/c小鼠前体T细胞,构建携带C57BL/6小鼠MHC-Ⅰ基因(H-2Db和H-2Kb)真核表达载体pIRES-H-2Db和pIRES-H-2Kb,分别转染BALB/c小鼠前体T细胞,构建分子嵌合前体T细胞。将分子嵌合前体T细胞回输BALB/c小鼠后7天,获取脾脏T淋巴细胞,与C57BL/6小鼠T细胞进行混合淋巴细胞培养,观测刺激指数(SI)。结果成功体外培养BALB/c小鼠前体T细胞,体外转染C57BL/6小鼠H-2Db和H-2Kb基因至BALB/c小鼠前体T细胞,H-2Db和H-2Kb蛋白表达率分别可达(14.90±0.56)%和(14.20±0.63)%。单向混合淋巴细胞培养显示,输注分子嵌合前体T细胞的BALB/c小鼠脾脏T细胞对C57BL/6小鼠T细胞SI,在pIRES-H-2Db和pIRES-H-2Kb转染的小鼠前体T细胞共注射组为(0.764±0.074),比空质粒组(0.983±0.081)和未转染组(0.994±0.142)明显下降(均P<0.05)。共注射组SI值分别与转染质粒pIRES-H-2Db组SI值(0.859±0.085)和转染质粒pIRES-H-2Kb组SI值(0.860±0.097)相比,差异均有统计学意义(均P<0.05)。结论输注分子嵌合MHC-Ⅰ基因前体T细胞的小鼠脾脏T细胞对异基因小鼠T细胞刺激反应明显减低。     

通过PD-1/PD-L1共刺激通路诱导小鼠胰岛移植免疫耐受的研究

目的 观察重组腺病毒Ad-PD-L1对诱导小鼠胰岛移植免疫耐受的作用.方法 酶切含有小鼠全长PD-L1 cDNA的pSport 1-mCD274质粒,亚克隆到穿梭质粒pShuale-GFP-CMV(-)上,再通过酶切、连接等方法构建腺病毒骨架质粒pAdxsi-GFP-CMV-PD-L1,转化DH5α感受态细菌,筛选阳性克隆.经酶切、测序鉴定正确,线性化后脂质体法转染293细胞进行包装、扩增,氯化铯密度梯度离心纯化病毒.链脲霉素(250 mg/kg)腹腔注射诱导C57BL/6(H-2b)小鼠糖尿病模型,将C57BL/6小鼠随机分为3组,A为对照组,B为Ad-EGFP处理组,C为Ad-PD-L1处理组,在进行胰岛移植的前1天,经尾静脉输注5×108 pfu的Ad-PD-L1.将DBA/2(H-2d)小鼠的胰岛移植在糖尿病小鼠肾被膜下.术后监测不同时间各组小鼠的血糖变化及胰岛移植物的存活时间,并观察混合淋巴细胞反应的特异性.结果 酶切及测序证实Ad-PD-L1构建成功.Ad-PD-L1在小鼠体内可以高效地表达PD-L1,Ad-PD-L1治疗组胰岛移植物的存活时间明显延长到27.63±3.51 d(P<0.01),混合淋巴细胞反应表明小鼠对供体反应特异性降低. 结论通过PD-L1/PD-1共刺激通路可以抑制T细胞的活化,从而延长了胰岛移植物的存活时间.     

携带VGLUT3-shRNA重组慢病毒的制备和鉴定

目的 制备携带谷氨酸转运体3( VGLUT3)基因小分子干扰RNA的重组慢病毒.方法 设计合成短发卡RNA (shRNA) VGLUT3对应的两条互补的寡核苷酸链,mU6-MCS-Ubi-EGFP质粒经Hpa Ⅰ和Xho Ⅰ进行双酶切与退火后的寡核苷酸连接,目的质粒转化感受态细胞,对克隆的菌落行聚合酶链反应(PCR)鉴定,再对PCR鉴定阳性的克隆进行测序,测序正确的重组质粒转染293细胞,同源重组产生Lentivirus-VGL UT3-shRNA并测定病毒滴度.结果 病毒滴度为1.5 × 109 TU/ml.结论 成功制备携带VGLUT3-shRNA的重组慢病毒.     

重组前强啡肽基因35型腺病毒载体的构建

目的 构建含前强啡肽(PDP)基因的重组35型腺病毒载体(Ad5/F35).方法 以pUC57-PDP重组质粒为模板扩增PDP基因,将回收的聚合酶链反应(PCR)产物片段克隆入pDC316载体,获得重组质粒pDC316-PDP.骨架质粒pBHG-fiberS/35和穿梭质粒pDC316-PDP共转染293细胞,同源重组产生Ad5/F35-PDP.经PCR鉴定目的 基因的表达.结果 PCR表明Ad5/F35-PDP质粒构建正确.结论 获得的Ad5/F35-PDP可以用于转基因治疗的实验研究.     

C57BL/6小鼠腹膜间皮细胞培养与鉴定

目的:建立C57BL/6小鼠腹膜间皮细胞(peritoneal-mesothelial-cells,PMCs)的体外培养方法。方法:选择8周龄C57BL/6雄性小鼠,0.25%胰蛋白酶-EDTA消化C57BL/6小鼠大网膜,细胞悬液经离心培养,进行形态学及细胞免疫组化鉴定。结果:分离培养的C57BL/6小鼠PMCs倒置显微镜下呈小圆形细胞,部分细胞聚集成小串葡萄状;72 h所有贴壁细胞均伸展,呈梭形等,边缘不整,细胞呈现拉网生长,与相邻的细胞相互连接;HE染色呈现多边形、椭圆形、短梭形,细胞浆染为淡红色;细胞免疫荧光组化鉴定上皮细胞标志分子E-cadherin及间充质标志分子Vimentin表达呈阳性,证实培养的细胞为PMCs。结论:C57BL/6小鼠腹膜间皮体外细胞培养成功,可为后续腹膜透析相关腹膜纤维化相关体外研究的开展提供可靠的细胞模型。     

门静脉输注供者脾细胞诱导的供者特异性免疫低反应性

目的 探讨经门静脉输注供者脾细胞能否诱导皮肤移植小鼠产生供者特异性的免疫低反应性及其可能机制.方法 取Balb/c小鼠,随机分为空白对照组(经小鼠门静脉输注RPMI 1640培养液)、受者脾细胞组(经小鼠门静脉输注Balb/c小鼠脾细胞)、供者脾细胞组(经小鼠门静脉输注C57BL/6小鼠脾细胞)、空白移植对照组(经小鼠门静脉输注RPMI 1640培养液,7 d后移植C57BL/6小鼠的皮肤)、实验对照组(经小鼠门静脉输注Balb/c小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)、实验组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C57BL/6小鼠的皮肤)以及第三方移植组(经小鼠门静脉输注C57BL/6小鼠脾细胞,7 d后移植C3H小鼠的皮肤).记录空白移植对照组、实验对照组、实验组和第三方移植组移植皮肤的存活时间,并观察移植皮肤的病理学变化;脾细胞输注后7 d,分别获取空白对照组、受者脾细胞组和供者脾细胞组小鼠的外周血、脾脏和肝脏,用流式细胞仪测定样本中CD4+CD25+Foxp3+调节性T淋巴细胞(CD4+CD25+Foxp3+Treg细胞)的比例.结果 实验组移植皮肤的存活时间为(19.8±4.6)d,明显长于空白移植对照组、实验对照组和第三方移植组,但仍未达到长期存活.皮肤移植后7 d,空白移植对照组和实验对照组的移植皮肤呈现重度急性排斥反应的病理学改变,而实验组移植皮肤呈现中度急性排斥反应的病理学改变.供者脾细胞组外周血、肝脏和脾脏中CD4+CD25+Foxp3+Treg细胞比例明显高于空白对照组和受者脾细胞组.结论 门静脉输注供者脾细胞可特异性地延长供者皮肤移植物的存活时间,减轻移植物的排斥反应,该效应可能与受者体内的CD4+CD25+Foxp3+Treg细胞增加有关.     

免疫磁珠两步法体外分离纯化小鼠脾脏CD4+CD25+调节性T细胞的应用研究

目的 研究免疫磁珠两步法体外分离纯化小鼠脾脏CD4+CD25+调节性T细胞(regulatory T cells,Treg)的效果.方法 将C57BL/6(H-2b)小鼠脾脏剪碎、过滤、去除红细胞后离心洗涤,获得小鼠脾细胞悬液.用Ficoll法分离获得单个核细胞后,通过免疫磁珠阴性加阳性选择两步法分离获得CD4+CD2...     

过表达膜联蛋白A1的人脂肪间充质干细胞治疗急性呼吸窘迫综合征小鼠的效果及其机制

目的探索过表达膜联蛋白A1(ANXA1)的人脂肪间充质干细胞(AMSC)治疗急性呼吸窘迫综合征(ARDS)小鼠的效果及其机制。方法采用实验研究方法。采用流式细胞术鉴定成人AMSC后, 取第3代细胞进行后续实验。采用随机数字表法(分组方法下同), 将细胞分为转染含ANXA1基因RNA序列的质粒的过表达ANXA1组、转染对应空载质粒的空载对照组, 另取细胞分为转染含ANXA1基因小干扰RNA序列的质粒的敲减ANXA1组、转染对应空载质粒的空载对照组, 转染后72 h, 于荧光显微镜成像系统下观察荧光表达情况, 分别采用蛋白质印迹法和实时荧光定量反转录PCR法检测ANXA1的蛋白和mRNA表达(样本数均为3)。取50只6～8周龄雄性C57BL/6J小鼠, 分为假伤组、单纯ARDS组、正常细胞组、过表达ANXA1组、敲减ANXA1组, 每组10只。将后4组小鼠均用内毒素/脂多糖制成ARDS肺损伤模型, 假伤组小鼠模拟致假伤。伤后即刻, 假伤组、单纯ARDS组小鼠均经尾静脉注射生理盐水, 正常细胞组、过表达ANXA1组、敲减ANXA1组小鼠对应注射正常AMSC、过表达ANXA1的AMSC、敲减A...     

Genetic analysis of glucose tolerance in inbred mouse strains. Evidence for polygenic control

To determine genetic factors involved in diabetes susceptibility in inbred strains of mice, we initially evaluated differences in fed plasma glucose and insulin concentrations among six strains (AKR/J, C3H/HeJ, C57BL/6J, C57L/J, DBA/2J, and SWR/J). There was considerable variation in fed plasma glucose concentration, with C3H/HeJ mice the most glucose tolerant (174 +/- 7 mg/dl) and C57BL/6J mice the least glucose tolerant (252 +/- 7 mg/dl, P less than .0001 vs. C3H/HeJ mice). Glycosylated hemoglobin of C57BL/6J mice (4.0 +/- 0.06%) was also higher than that of C3H/HeJ mice (3.52 +/- 0.06%, P less than .0001). The fed plasma insulin concentration did not differ between these two strains. Glucose tolerance was further evaluated in overnight-fasted C3H/HeJ and C57BL/6J mice by an intraperitoneal glucose tolerance test (IPGTT). Although fasting plasma glucose did not differ, the most remarkable difference in plasma glucose during IPGTT between C57BL/6J and C3H/HeJ mice was noted at 30 min (489 +/- 29 vs. 227 +/- 20 mg/dl, P less than .001). To determine the number of genes involved in the phenotypic difference in glucose tolerance, C57BL/6J males were crossed with C3H/HeJ females (F1, C3H/HeJ X C57BL/6J), and the F1 hybrid females were backcrossed with C57BL/6J males (backcrossed, F1 X C57BL/6J). Plasma glucose after 30 min on IPGTT was 219 +/- 8 (n = 21), 456 +/- 18 (n = 23), and 292 +/- 13 (n = 23) mg/dl for C3H/HeJ, C57BL/6J, and F1 mice, respectively (P less than .001 for all comparisons).(ABSTRACT TRUNCATED AT 250 WORDS)     

胚胎干细胞移植修复脊髓损伤的实验研究

目的观察胚胎干细胞（embryonic stem cell,ES）诱导的神经前体细胞移植,对小鼠脊髓损伤神经功能恢复的影响。方法取由上海市发育生物学重点实验室提供的ES进行细胞培养和体外诱导,收集ES衍生细胞。并进行RT-PCR检测。将50只C57/BL6J小鼠制备为T9、10脊髓半横断模型,将存活的28只小鼠随机分为三组。假手术组（A组）：9只,未作任何处理;手术/细胞组（B组）：10只,于距损伤区域以远约1cm的椎管内注射2~3μl制备的ES衍生细胞,总细胞数为9×105个;手术/DMEM组（C组）：9只,按B组方法注射2~3μl DMEM。术后1、2、4、6和8周采用BBB后肢功能评分观察小鼠神经功能恢复情况,取损伤脊髓进行X-gal染色和免疫组织化学染色观察。结果ES经体外诱导培养,呈圆形或椭圆形小集落生长,有1个或多个核仁。RT-PCR检测,ES细胞诱导后表达巢蛋白及微管相关蛋白,但未表达胶质纤维酸性蛋白。小鼠实验,BBB后肢功能评分显示术后各时间点A组与B、C组比较,差异均有统计学意义（P〈0.01）。B组与C组比较,1、2和4周时,差异有统计学意义（P〈0.01）;6、8周时,组间差异无统计学意义（P〉0.05）。X-gal染色观察,B组呈阳性染色,A、C组均为阴性。免疫组织化学染色观察,B组在损伤脊髓部位,表达兔抗神经微丝蛋白,未表达胶质纤维酸性蛋白。结论将ES培养诱导分化为神经前体细胞移植后,能够存活、迁移,并分化为神经元,但未明显改善神经功能。     

Evidence that testosterone regulates Leydig cell 3 beta-hydroxysteroid dehydrogenase-isomerase activity by a trans-acting factor distal to the androgen receptor.

Leydig cells are a target for their own steroid product, testosterone, and thus could be subject to short-loop feedback regulation by androgens. The authors previously reported that 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta HSD) activity was higher in freshly isolated Leydig cells from C57BL/6J than those from C3H/HeJ inbred mice. To determine whether this strain-related difference in 3 beta HSD activity could be mediated by differential sensitivity to feedback effects of testosterone, Leydig cells from the two strains were cultured in the presence or absence of testosterone, the synthetic androgen receptor agonist, mibolerone, or the nonaromatizable androgen, dihydrotestosterone. After 7 days of treatment, all three androgens significantly decreased 3 beta HSD activity in Leydig cells from C57BL/6J, but not from C3H/HeJ mice. When Leydig cells were cultured with hydroxyflutamide, an androgen receptor antagonist, the effect of testosterone was negated. To determine whether the strain-related difference in sensitivity to testosterone was mediated by a difference in the androgen receptor protein, Leydig cells from reciprocal F1 hybrid lines of mice were cultured in the presence or absence of testosterone. Testosterone treatment inhibited 3 beta HSD activity in both F1 lines to the same extent as observed for Leydig cells from C57BL/6J mice. Thus, there is a strain-related difference in the response to testosterone, but it cannot account for the strain-related difference in Leydig cell 3 beta HSD activity because the high 3 beta HSD strain (C57BL/6J) is the sensitive strain. Although the effect on C57BL/6J Leydig cells is androgen receptor-mediated, the dominant effect of testosterone on both F1 lines rules out a difference in the androgen receptor protein per se. However, the data are consistent with the difference being in a trans-acting factor distal to the androgen receptor.     

Effects of Thy-1+ cell depletion on the capacity of donor lymphoid cells to induce tolerance across an entire MHC disparity in sublethally irradiated adult hosts

Thy-1+ cell depletion with anti-Thy-1.2 mAb and complement markedly reduced the capacity of C57BL/6J, H-2b bone marrow to establish mixed lymphoid chimerism and induce tolerance to C57BL/6J skin grafts across an entire MHC disparity in BALB/c, H-2d hosts conditioned with sublethal, fractionated 7.5 Gy total-body irradiation. In this model tolerance can be transferred to secondary irradiated BALB/c hosts only by cells of C57BL/6J donor, not host, genotype isolated from the spleens of tolerant hosts. Thy-1+ cell depletion abolished the capacity of C57BL/6J donor cells from tolerant BALB/c host spleens to transfer tolerance. The capacity of semiallogeneic BALB/c x C57BL/6J F1, H-2d/b donor BM and spleen cells to induce chimerism and tolerance to C57BL/6J skin grafts in BALB/c parental hosts was also reduced by Thy-1+ cell depletion. Thus the requirement for donor Thy-1+ cells cannot be explained simply on the basis of alloaggression. It is unlikely that the requisite Thy-1+ cells are nonspecific suppressor cells: Thy-1+ cell depletion had no effect on the slight but significant prolongation of third-party C3H/HeJ, H-2k skin grafts in irradiated BALB/c hosts injected with allogeneic C57BL/6J or semiallogeneic BALB/c x C57BL/6J F1 BM compared to irradiated controls injected with medium only. Furthermore, injections of semiallogeneic F1 spleen cells had no significant effect on the survival of the third-party grafts, although these cells were fully capable of inducing tolerance, and their capacity to induce tolerance was significantly reduced by Thy-1+ cell depletion. The requirement for a specific population of lymphoid cells, i.e. Thy-1+, remains unexplained but suggests that donor cells might play a role in the induction or maintenance of tolerance in this model other than merely providing a circulating source of donor antigens.     

Up-Regulation of Donor Dendritic Cell PD-L1 Expression Reduced Recipient Lymphocyte Activation and Proliferation In Vitro

ObjectiveTo observe the effects of dendritic cells (DC) in donor C57BL/6 (H-2^b) micetransfected with recombinant adenovirus vector Ad-PD-L1 on proliferation and activation of lymphocytes in recipient DBA/2 (H-2^d) mice.MethodsThe pSport 1-mSD274 plasmid containing the full-length PD-L1 cDNA of the mouse was digested and subcloned to the shuttle plasmid pShuttle-GFP-CMV(-), and then the adenovirus skeleton plasmid pAdxsi-GFP-CMV-PD-L1 was constructed by enzymolysis and ligation, transformed into DH5α sensitive bacteria, and screened for positive clones. After enzyme digestion, sequencing, and identification, 293 cells were transfected with liposome after linearization for packaging and amplification, and the virus was purified by cesium chloride density gradient centrifugation. DC of donor C57BL/6 mice were isolated, cultured, and divided into the following 3 groups: group A, adenovirus vector Ad-PD-L1 transfection group; group B, empty vector transfection group; and group C, control group. Western blot was used to detect the expression of PD-L1 in each group of cells after transfection. Isolate lymphocytes from recipient DBA/2 mice were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and mixed with DC of donor C57BL/6 mice with lymphocytes of recipient DBA/2 mice. Flow cytometry was performed to observe the proliferation of lymphocytes.ResultsDigestion and sequencing confirmed that the recombinant adenovirus vector Ad-PD-L1 containing PD-L1 was successfully constructed. After transfection with DC of donor C57BL/6 mice, the expression of PD-L1 increased by 37% (P < .05), and the PD-L1 transfected DC and recipient DBA/2. Mouse lymphocytes were cocultured. Compared with the control group, the increased expression of PD-L1 significantly inhibited the proliferation and activation of lymphocytes. The lymphocyte proliferation of DBA/2 mice decreased by 41% (P < .01).ConclusionThe recombinant adenovirus vector Ad-PD-L1 containing the mouse PD-L1 gene was successfully constructed. After transfection with dendritic cells of donor C57BL/6 mice, PD-1/PD-L1 inhibited lymphocytes proliferation and activation of recipient DBA/2 mice through costimulatory pathway.     

Chimaerism in lymph nodes of F1 into irradiated parental recipient chimaeras rejecting skin allografts from the other parent.

Mice of the C57BL strain were irradiated with 800 R over the whole body. The next day they received i.v. a mixture of 50 x 10(6) spleen and bone marrow cells from (C57BL x CBA-T6T6)F1 hybrid mice, and were challenged with CBA-T6T6 skin grafts later on. About 20% of the recipients rejected the CBA-T6T6 skin, whereas the others were completely tolerant for more than 200 days. By using the cytotoxic test, we found that both tolerant and nontolerant recipients were complete chimaeras, i.e., had only (C57BL x CBA-T6T6)F1 cells in their lymph nodes. However, analysis of the same mice by the chromosome marker technique disclosed a proportion of host (C57BL) cells in lymph nodes of both tolerant and nontolerant chimaeras. The percentage of host metaphases in nontolerant chimeras was significantly higher than that in tolerant chimaeras (P less than 0.01). It is possible therefore that the CBA-T6T6 skin grafts were rejected by residual host (C57BL) cells rather than (C57BL x CBA-T6T6)F1 cells reacting against skin-specific transplantation antigen(s) of the parental graft.     

Specific inhibition of the graft-versus-host reaction in fetal or neonatal liver chimeras.

The fetal liver chimera system was used as a model to study the nature of transplantation tolerance in radiation chimeras. A permanent state of tolerance was induced in (C3H/eb times C57BL/6)F1 irradiated mice after reconstitution with parental C57BL/6 fetal or neonatal liver cells. It was found that although enough host hemopoietic cells were present in such chimeras to provide antigenic stimulation, subsequent inoculation of these chimeras with C57BL/6 immunocompetent cells syngeneic to liver donor cells specifically abolished their response against the host. In addition, cells obtained from liver chimeras after their challenge with C57BL cells were unable to produce a graft-versus-host response upon transfer to (C3H/eb times C57BL/6)F1 newborn mice. Transfer of serum of these mice could not prevent immune reactivity of syngeneic C57BL/6 cells neither in the graft-versus-host nor in the mixed lymphocyte culture assays. These observations are compatible with the hypothesis that suppressor cells may differentiate within the fetal liver, which specifically inhibits the immune reactivity of syngeneic cells, and thus leads to the establishment of tolerance.     

Thy 1+ donor cells that promote allograft tolerance in sublethally irradiated MHC-disparate hosts.

We have previously demonstrated that depletion of Thy 1+ cells impairs the capacity of C57BL/6J, H-2b bone marrow cells, BMC, and BALB/cxC57BL/6J F1 BMC and spleen cells (SC) to establish mixed lymphoid chimerism and tolerance for donor-specific skin grafts in sublethally irradiated (240 cGy x3) BALB/c, H-2d hosts. In the present studies incubation with anti Ly2.2 + C markedly reduced the capacity of BALB/cxC57BL/6J, F1 SC to induce tolerance and chimerism (P less than .001). Incubation with anti-L3T4 + C had an inhibitory effect of borderline significance (P less than .04). Incubation with L-leucyl-L-leucine methyl ester (which removes NK cells, Tc, and precursor Tc) had no effect on the capacity of either C57BL/6J BMC or BALB/cxC57BL/6J F1 BMC or SC to establish chimerism and induce skin graft tolerance. These results suggest that tolerance-promoting Thy 1+ cells are not cytotoxic T cells. Both Ly2+ noncytotoxic CD8+ and L3T4+ noncytotoxic CD4+ cells may be involved. Alternatively the requisite Thy 1+ cells may be immature T cells that express both Ly2 and L3T4.     

外源性骨髓细胞在体诱导形成毛囊上皮细胞的研究

目的 观察外源性骨髓细胞能否在体诱导形成皮肤的上皮细胞.方法 获取转增强型绿色荧光蛋白基因的C57BL/6J小鼠原代骨髓细胞和野生型C57BL/6J小鼠原代表皮干细胞,植入野生型C57BL/6J小鼠背部创面并打包.分A、B两组各10只,A组每只植入1.0×107个单纯骨髓细胞,B组每只植入混合骨髓细胞、表皮干细胞各1.0×107个.创面痊愈后应用荧光法和免疫组织化学技术检测新生皮肤内EGFP的表达.结果 两组动物3个月后创面痊愈并有完整的毛发生长.A组新生皮肤内未见EGFP的表达;B组所有新生皮肤(100%)均可见EGFP阳性细胞群,多分布在毛囊内.结论 创伤后小鼠毛囊的再生过程中,骨髓细胞-表皮干细胞接触诱导对骨髓来源细胞向毛囊上皮细胞转化至关重要.