Exonuclease-1缺失延缓端粒酶缺失小鼠造血微环境的衰老

目的研究Exo-1对端粒酶缺失小鼠造血微环境衰老的影响。方法以端粒酶基因敲除小鼠（Terc-/-）和Exo-1基因敲除小鼠（Exo-1-/-）杂交,并进一步互交产生第三代端粒酶基因敲除小鼠（G3Terc-/-）以及第三代Terc和Exo-1双基因敲除小鼠（G3Terc-/-Exo-1-/-）。以CD45.1野生型小鼠的骨髓细胞为供体,以2月龄G3Terc-/-或G3Terc-/-Exo-1-/-小鼠为受体,进行骨髓移植。在受体小鼠9月龄时,取骨髓、脾脏、胸腺、外周血等组织器官的细胞进行流式分析,研究G3Terc-/-和G3Terc-/-Exo-1-/-受体小鼠中的野生型供体来源的造血干细胞的发育分化。结果同G3Terc-/-小鼠相比,G3Terc-/-Exo-1-/-双基因敲除受体小鼠骨髓中野生型供体来源的B220＋细胞比例升高,前体B细胞的比例也明显升高;脾脏B220＋细胞的比例明显升高;胸腺发育正常;外周血中B220＋细胞比例升高。结论 Exo-1缺失延缓了端粒酶基因敲除小鼠造血系统微环境的衰老,从而逆转了端粒功能障碍引起的骨髓造血干细胞发育分化异常。     

p18INK4C基因缺失增强造血干细胞在亚致死剂量照射小鼠体内长期植入能力

观察p18INK4C(p18)基因缺失对造血干细胞(HSC)在亚致死剂量照射小鼠体内长期植入的影响. 供体为p18基因缺失型(p18(/()纯系C57BL/6小鼠(CD45.2表型), 竞争性细胞来源于C57BL/6-Ly5.1(CD45.1/2)双表型小鼠, 受体为野生型(p18+/+)C57BL/6-Ly5.1(CD45.1)小鼠. 竞争性骨髓移植(cBMT)实验根据受体小鼠照射剂量的不同分为3个剂量组(10 Gy, 5 Gy和1 Gy). 供体细胞和竞争性细胞1:1混合后移植, 移植后采集外周血和骨髓细胞用流式细胞仪检测各细胞比例. 造血恢复移植实验: 移植后检测外周血白细胞计数评价移植后造血恢复速度. 10和5 Gy照射剂量组, 供体细胞和竞争性细胞成功植入, 而1 Gy照射剂量组无供体细胞植入. 无论在10 Gy或是5 Gy照射剂量情况下, 供体细胞在受体内的比例均高于竞争性细胞. 移植后6周, 10和5 Gy照射剂量时外周血中供体细胞比例分别为竞争性细胞的1.46±0.21倍和1.64±0.43倍, 14周时分别为竞争性细胞的1.84±0.25倍和2.00±0.49倍, 26周时分别为竞争性细胞的3.13±0.79倍和3.24±1.33倍. 移植后6个月, 10 Gy照射剂量时骨髓细胞中供体细胞比例为竞争性细胞的7.68±4.42倍, 5 Gy照射剂量时为竞争性细胞的10.83±2.98倍. 移植后6个月, 在10和5 Gy照射剂量组之间骨髓中造血细胞植入率相当, 分别为(85.53±8.71)%和(80.87±2.87)% (P = 0.457). p18(/(细胞与p18+/+细胞相比, 移植后造血恢复的速度相当. p18基因缺失可以显著增强HSC在亚致死剂量照射小鼠体内的长期植入能力.     

p18INK4C基因缺失增强造血干细胞在亚致死剂量

观察p18^INK4C (p18)基因缺失对造血干细胞(HSC)在亚致死剂量照射小鼠体内长期植入的影响. 供体为p18基因缺失型(p18^-/-)纯系C57BL/6小鼠(CD45.2表型), 竞争性细胞来源于C57BL/6-Ly5.1(CD45.1/2)双表型小鼠, 受体为野生型(p18^+/+)C57BL/6-Ly5.1(CD45.1)小鼠. 竞争性骨髓移植(cBMT)实验根据受体小鼠照射剂量的不同分为3个剂量组(10 Gy, 5 Gy和1 Gy). 供体细胞和竞争性细胞1:1混合后移植, 移植后采集外周血和骨髓细胞用流式细胞仪检测各细胞比例. 造血恢复移植实验: 移植后检测外周血白细胞计数评价移植后造血恢复速度. 10和5 Gy照射剂量组, 供体细胞和竞争性细胞成功植入, 而1 Gy照射剂量组无供体细胞植入. 无论在10 Gy或是5 Gy照射剂量情况下, 供体细胞在受体内的比例均高于竞争性细胞. 移植后6周, 10和5 Gy照射剂量时外周血中供体细胞比例分别为竞争性细胞的1.46±0.21倍和1.64±0.43倍, 14周时分别为竞争性细胞的1.84±0.25倍和2.00±0.49倍, 26周时分别为竞争性细胞的3.13±0.79倍和3.24±1.33倍. 移植后6个月, 10 Gy照射剂量时骨髓细胞中供体细胞比例为竞争性细胞的7.68±4.42倍, 5 Gy照射剂量时为竞争性细胞的10.83±2.98倍. 移植后6个月, 在10和5 Gy照射剂量组之间骨髓中造血细胞植入率相当, 分别为(85.53±8.71)%和(80.87±2.87)% (P = 0.457). p18^-/-细胞与p18^+/+细胞相比, 移植后造血恢复的速度相当. p18基因缺失可以显著增强HSC在亚致死剂量照射小鼠体内的长期植入能力.     

小鼠脑组织腺苷A2A受体缺失模型的建立与评价

制备了脑组织腺苷A2A受体基因缺失的小鼠模型并对该模型进行评价。在采用2次6.2 Gy X线间隔照射对敲除A2A受体基因的雌性C57BL/6小鼠进行清髓处理后, 将野生型雄性C57BL/6小鼠骨髓细胞移植到其体内, 使其脑组织的A2A受体仍保持缺失型。然后对移植效果进行鉴定, 并对模型的生理指标进行观察和评价。结果发现: 骨髓移植6周后, 受体小鼠的白细胞性染色体基因由雌性变为雄性; 骨髓中A2A受体阳性细胞率为94.85%, 而脑内A2A受体mRNA与A2A受体基因敲除小鼠比较, 无显著差异。模型小鼠除心率略低于野生型小鼠外, 在呼吸频率、脑含水量以及脑内谷氨酸含量等生理指标上均与野生型小鼠和A2A基因敲除小鼠无显著差异。     

造血干细胞移植条件对小鼠造血重建的影响

通过同种基因型小鼠构建造血干细胞移植模型,将预处理的全骨髓单个核细胞或c-Kit⁺造血干细胞移植至致死剂量照射的受体小鼠体内,动态监测移植2～16周后受体小鼠体内供体来源细胞造血重建以及嵌合情况,以期揭示不同群体的供体细胞以及预处理等因素对小鼠造血干细胞移植后造血重建的影响。实验结果显示,移植后早期（2周）全骨髓单个核细胞组髓系比例要高于c-Kit⁺细胞移植组,但全骨髓移植组受体小鼠呈现出较大的移植后不良反应,出现脱毛、食欲不振以及体重减轻的症状。c-Kit⁺细胞移植组在淋系重建上要早于全骨髓移植组,供体细胞的嵌合植入也早于全骨髓移植组,但两组实验组最终均能完成造血重建过程。实验结果表明c-Kit⁺细胞移植组在移植后能够较快地实现供体细胞植入,进而开始造血重建,且c-Kit⁺ 细胞移植组的不良反应要低于全骨髓移植组。结果说明在整体造血重建效果上c-Kit⁺细胞移植组要优于全骨髓移植组。     

小鼠脑组织腺苷A2A受体缺失模型的建立与评价

制备了脑组织腺苷A2A受体基因缺失的小鼠模型并对该模型进行评价。在采用2次6.2 Gy X线间隔照射对敲除A2A受体基因的雌性C57BL/6小鼠进行清髓处理后, 将野生型雄性C57BL/6小鼠骨髓细胞移植到其体内, 使其脑组织的A2A受体仍保持缺失型。然后对移植效果进行鉴定, 并对模型的生理指标进行观察和评价。结果发现: 骨髓移植6周后, 受体小鼠的白细胞性染色体基因由雌性变为雄性; 骨髓中A2A受体阳性细胞率为94.85%, 而脑内A2A受体mRNA与A2A受体基因敲除小鼠比较, 无显著差异。模型小鼠除心率略低于野生型小鼠外, 在呼吸频率、脑含水量以及脑内谷氨酸含量等生理指标上均与野生型小鼠和A2A基因敲除小鼠无显著差异。     

大剂量~(60)Co照射后造血干细胞潜在的残留损伤

本文对受到临界全致死剂量——8.5Gy~(60)Co照射后的小鼠造血干细胞(HSC)的自我更新力进行了测试,并对照射后4个月,造血功能业已恢复的小鼠HSC的造血重建功能进行了研究。结果表明:应用骨髓连续移植实验所测得的照后3个月中骨髓CFU_s的自我更新潜能明显衰退了。用照射后造血恢复小鼠的CFU_s给全致死剂量照射的受体进行移植治疗,发现其移植效力比正常的显著减弱。在受体存活30天时,CFU_s的再生速率只有正常的1/17。通过对性染色体追踪观察的资料分析,此种小鼠的??造血细胞在受体中难以形成长期稳定的嵌合体。以上事实反映出,照射后小鼠的HSC的造血重建功能大大削弱了,揭示出残存干细胞质量上的缺陷。对于这种潜在的残留损伤的机制,从“干细胞增殖力耗竭学说”和“基因自我修整假说”的角度进行了讨论。     

胎肝造血干细胞的移植特性

胚胎发育中,肝脏是一个重要的造血器官。近年来胎肝移植的临床应用重新引起了人们的关注。本文应用染色体的 C-带染色法研究了小鼠骨髓和胎肝造血干细胞在照射受体小鼠中的增殖能力与相互间的竞争作用。实验结果表明胎肝造血干细胞在成年骨髓中的植入率比较同样条件下的成年骨髓造血干细胞低,但胎肝造血干细胞比较成年骨髓造血干细胞具有更强的自我更新或增殖能力。在同种胎肝造血干细胞移植中,为了降低同种移植抗力,提高移植的胎肝造血干细胞在受体中的耐受性,移植前对受体作适当的免疫抑制处理是必要的。因此,克服个体发育屏障和移植免疫屏障是提高同种胎肝造血干细胞移植效果中两个重要的研究课题。     

RunX3对小鼠骨髓造血干细胞功能的影响

目的研究RunX3基因对造血干细胞自我更新和分化能力的影响。方法流式细胞术测定小鼠骨髓干细胞和外周血单个核细胞的比例;通过竞争性骨髓移植实验检测RunX3转基因小鼠骨髓干细胞的功能。结果移植后来源于RunX3-/-小鼠骨髓干细胞供体的外周血细胞占总外周血细胞的比例与野生对照鼠相比无明显差异,移植后来源于RunX3-/-小鼠骨髓干细胞供体的外周血中髓系细胞占总外周血髓系细胞的比例较野生型对照鼠高。结论RunX3基因缺失对骨髓造血干细胞的自我更新没有影响,但其可能参与了骨髓造血干细胞的分化过程。     

胎肝造血干细胞在扩散盒培养条件下移植特性的变化

经体内扩散盒培养6d 后的 LACA 小鼠胎肝细胞移植给照射的同系成年小鼠,造血干细胞在受体脾脏和骨髓中的有效植入率比正常胎肝细胞明显提高。但这种效果在同种异基因受体小鼠中则完全消失。实验结果表明,个体发育屏障和移植免疫屏障是决定同种胎肝移植能否成功的两个重要因素。胎肝细胞经体内或体外培养后可以模拟造血干细胞在体内的发育成熟,从而增强对成年造血微环境的适应性。用短期体内培养的方法,可以改变胎肝造血干细胞的某些生理特性,从而减弱个体发育屏障,但不能克服胎肝同种移植中的免疫性抗力。为了保证同种胎肝移植的成功,必须进一步同时克服两种屏障。     

Telomerase deficiency impairs differentiation of mesenchymal stem cells

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR(-/-)) and wild-type (WT) mice. The MSCs isolated from mTR(-/-) mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR(-/-)MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation.     

Role of interferon-gamma in the evolution of murine bleomycin lung fibrosis

IFN-gamma production is upregulated in lung cells (LC) of bleomycin-treated C57BL/6 mice. The present study characterizes the time course, cellular source, and regulation of IFN-gamma expression in bleomycin-induced lung injury. IFN-gamma mRNA in LC from bleomycin-treated mice peaked 3 days after intratracheal instillation. IFN-gamma protein levels were increased at 6 days, as was the percentage of LC expressing IFN-gamma. CD4+, CD8+, and natural killer cells each contributed significantly to IFN-gamma production. IL-12 mRNA levels were increased at 1 day in LC of bleomycin-treated mice. Anti-IL-12 and anti-IL-18 antibodies decreased IFN-gamma production by these cells. To define the role of endogenous IFN-gamma in the evolution of bleomycin lung injury, we compared the effect of bleomycin in mice with a targeted knockout mutation of the IFN-gamma gene (IFN-gamma knockout) and wild-type mice. At 14 days after intratracheal bleomycin, total bronchoalveolar lavage cell counts and lung hydroxyproline were decreased in IFN-gamma knockouts compared with wild-type animals. There was no difference in morphometric parameters of fibrosis. Our data show that enhanced IFN-gamma production in the lungs of bleomycin-treated mice is at least partly IL-12 and IL-18 dependent. Absence of IFN-gamma in IFN-gamma knockout mice does not increase pulmonary fibrosis. Endogenous IFN-gamma may play a proinflammatory or profibrotic role in bleomycin-induced lung fibrosis.     

Generation of mice by transplantation of an adult spermatogonial cell line after cryopreservation

Objectives:  The key to fertility in adult males is production of mature spermatogenic cells. Spermatogonial stem cells (SSC) have the dual capacity of self-renewal and of differentiation into mature sperm. SSC transplantation may provide potential treatment for specific male infertilities. However, until now, there has been no evidence of offspring produced by transplantation of adult SSC line cells in humans or other mammals.
Materials and methods:  A new line of SSCs from adult C57BL/6 mouse was established by using magnetic-activated cell sorting. The cell line was characterized by immunocytochemistry, karyotype analysis and telomeric repeat amplification protocol (TRAP) telomerase activity assay. Spermatogenic function was examined by allograft into germ cell-ablated recipient mice.
Results:  For more than 14 months with more than 65 maintenance passages, the cell line showed a normal karyotype (40, XY) and high telomerase activity. It represented a Thy-1+, Oct4+, SSEA-1-, c-kit- (99 ± 1%) cell subpopulation. We cryopreserved these SSCs and successfully produced normal offspring after transplanting them into testes of busulphan-sterilized mice.
Conclusions:  We established and long-term maintained an adult SSC line with normal spermatogenic function, without the need of genetic modification; thus, this study provides a model system for basic research and clinical application.     

Regeneration of the exocrine pancreas is delayed in telomere-dysfunctional mice

Introduction

Telomere shortening is a cell-intrinsic mechanism that limits cell proliferation by induction of DNA damage responses resulting either in apoptosis or cellular senescence. Shortening of telomeres has been shown to occur during human aging and in chronic diseases that accelerate cell turnover, such as chronic hepatitis. Telomere shortening can limit organ homeostasis and regeneration in response to injury. Whether the same holds true for pancreas regeneration in response to injury is not known.

Methods

In the present study, pancreatic regeneration after acute cerulein-induced pancreatitis was studied in late generation telomerase knockout mice with short telomeres compared to telomerase wild-type mice with long telomeres.

Results

Late generation telomerase knockout mice exhibited impaired exocrine pancreatic regeneration after acute pancreatitis as seen by persistence of metaplastic acinar cells and markedly reduced proliferation. The expression levels of p53 and p21 were not significantly increased in regenerating pancreas of late generation telomerase knockout mice compared to wild-type mice.

Conclusion

Our results indicate that pancreatic regeneration is limited in the context of telomere dysfunction without evidence for p53 checkpoint activation.     

DDR2基因缺失对小鼠骨髓间充质干细胞生物学特性的影响

目的:骨髓间充质干细胞(Bone Mesenchymal Stem Cells,BMSCs)是骨再生工程中重要的种子细胞,它对骨组织缺损的修复有着良好的效果。但是BMSCs向成骨细胞分化并修复骨组织缺损是是由细胞外因子共同作用产生的结果。DDR2(Discoidin Domain Receptor 2)作为I型胶原的特异性受体在成骨细胞的分化中发挥重要的调节作用。而对于其在BMSCs向成骨细胞的分化过程中的所起到的作用还鲜有研究,对其作用机理尚不明确。因此我们希望通过分离、培养并鉴定比较DDR2基因缺失小鼠与野生型小鼠来源的骨髓间充质干细胞了解其生物学特性,为后续的实验奠定理论基础。方法:采用改良型的全骨髓贴壁细胞分离方法分离培养两种小鼠来源的骨髓间充质干细胞,采用流式细胞技术鉴定其表面标记物的表达,并利用诱导培养液诱导骨髓间充质干细胞向成骨细胞和成脂肪细胞分化。结果:分离培养的两种骨髓间充质干细胞形态一致,增殖能力和自我更新能力强,流式细胞术检测其表面标记物CD29,Sca-1均表达阳性,CD105,CD45表达为阴性,分离得到的两种细胞均有向成骨细胞和成脂肪细胞分化的能力,但可以明显观察到DDR2基因缺失小鼠的骨髓间充质干细胞的成骨分化能力减弱。结论:本实验通过对于DDR2基因缺失小鼠BMSCs分离、培养和鉴定,初步探索DDR2基因缺失在在成骨过程中的作用结果,为进一步研究提高BMSCs的成骨分化能力奠定理论基础。经实验证明,DDR2基因缺失小鼠来源的骨髓间充质干细胞虽然仍具备干细胞的生物学特性,但其向成骨细胞的分化能力明显减弱,说明DDR2基因缺失对其骨髓间充质干细胞的成骨分化等有着重要的影响。     

黏蛋白1基因敲除小鼠模型的构建

黏蛋白1(MUC1)属黏蛋白家族成员,分布于上皮细胞膜表面,由于在免疫炎症反应以及肿瘤发生中的重要作用而日益受到重视.为了进一步深入研究MUC1的生物学功能,构建了Muc1基因敲除小鼠模型.首先,根据小鼠Muc1基因组序列设计基因剔除策略,将2个loxP位点分别插在外显子2和3两侧,构建基因剔除载体Muc1-ABRLFn-pBR322.以电穿孔方法将载体导入胚胎干细胞(ES细胞),用G418和更昔洛韦进行正负筛选获得4个同源重组的ES细胞克隆.挑选其中一个阳性ES克隆行囊胚显微注射,获得16只嵌合率大于50%的雄鼠;其次,利用嵌合雄鼠与C57BL/6J野生型雌鼠交配后获得11只floxP杂合子小鼠(10雄1雌),通过杂合子小鼠回交,并进一步与EⅡa-Cre小鼠交配,最终成功得到Muc1全身敲除小鼠,其中纯合子小鼠未出现胚胎致死现象.初步表型观察未发现Muc1基因敲除相关器官组织结构的异常改变.本研究为MUC1的生物学功能的挖掘,尤其是MUC1在肿瘤发生转移中的作用机制的揭示提供了实验平台.     

Generation of mice with hepatocyte-specific conditional deletion of sphingosine kinase 1

SphK1 gene has different roles in various types of cells in liver diseases, but most studies are based on global knockout mice, which hampers the study on the cellular and molecular mechanisms of SphK1. In order to further study the role of SphK1 in liver, SphK1 conditional knockout mice were constructed. A liver-specific SphK1 gene knockout mouse model was constructed by the Cre/Loxp recombinant enzyme system. PCR technologies and western blotting were used to identified the elimination of SphK1 gene in hepatocytes. SphK1^flox/flox mice were used as a control group to verify the effectiveness of SphK1 liver-specific knockout mice from the profile, pathology, and serology of mice. The ablation of SphK1 in hepatic parenchymal cells was demonstrated by fluorescent in situ hybridization and the contents of S1P and Sph were measured by ELISA kit. The genotypes of liver in SphK1 conditional knockout mice were different from that of other organs. The mRNA and protein levels of SphK1 in liver tissue of SphK1 conditional knockout mice were almost depleted by compared with SphK1^flox/flox mice. Physiology and pathology showed no significant difference between SphK1 liver conditional knockout mice and SphK1^flox/flox mice. Additionally, SphK1 was eliminated in hepatocytes, leading to the reduce of S1P content in hepatocytes and liver tissues and the increase of Sph content in hepatocytes. The model of SphK1 gene liver conditional knockout mice was successfully constructed, providing a tool for the study of the roles of SphK1 in hepatocyte and liver diseases.