Induction of embryonic stem cells to hematopoietic cellsin vitro

In order to get hematopoietic cells from embryonic stem (ES) cells and to study development mechanisms of hematopoietic cells, the method of inducing embryonic stem cells to hematopoietic cells was explored by differenciating mouse ES cells and human embryonic cells in three stages. The differentiated cells were identified by flow cytometry, immunohistochemistry and Wright’s staining. The results showed that embryoid bodies (EBs) could form when ES cells were cultured in the medium with 2-mercaptoethanol (2-ME). However, cytokines, such as stem cell factor (SCF), thrombopoietin (TPO), interleukin-3 (IL-3), interleukin-6 (IL-6), erythropoietin (EPO) and granular colony stimulating factor (G-CSF), were not helpful for forming EBs. SCF, TPO and embryonic cell conditional medium were useful for the differentiation of mouse EBs to hematopoietic progenitors. Eighty-six percent of these cells were CD34⁺ after 6-d culture. Hematopoietic progenitors differentiated to B lymphocytes when they were cocultured with primary bone marrow stroma cells in the DMEM medium with SCF and IL-6. 14 d later, most of the cells were CD34^－CD38⁺. Wright’s staining and immunohistochemistry showed that 80% of these cells were plasma-like morphologically and immunoglubolin positive. The study of hematopoietic cells from human embryonic cells showed that human embryonic cell differentiation was very similar to that of mouse ES cells. They could form EBs in the first stage and the CD34 positive cells account for about 48.5% in the second stage.     

Effect of tramadol on immune responses and nociceptive thresholds in a rat model of incisional pain

Objective： To evaluate the effects oftramadol on the proinflammatory responses in a rat model of incisional pain by investigating its effects on nociceptive thresholds and serum interleukin-6 （IL-6） and IL-2 levels. Methods： Forty-two male Sprague-Dawley （SD） rats scheduled for plantar incision were randomly divided into 7 groups 01=6 in each group）. Rats in Group 1 receiving general anesthesia with no incision were served as control; At 30 min before skin incision, Groups 2-5 were given 5 ml normal saline or 1, 10, and 20 mg/kg tramadol, respectively, intraperitoneally （i.p.）; Group 6 received 10 mg/kg tramadol after operation; Group 7 received 10 mg/kg tramadol before incision, followed by 200 μg/kg naloxone after operation. Mechanical allodynia was measured by electronic yon Frey filament to evaluate the nociceptive thresholds 1 h before incision, and 1 h and 2 h after operation. Serum IL-6 and IL-2 levels were measured by enzyme-linked immunosorbent assay （ELISA） 2 h after operation. Results： Mechanical thresholds decreased significantly and serum IL-6 level increased significantly after operation in Group 2 compared with control （P〈0.01）, and these changes were reversed respectively by tramadol in a dose-dependent manner （P〈0.05 and P〈0.01, respectively）. IL-2 level remained unchanged after operation in Group 2, but decreased in Group 3 （P〈0.05）, then gradually returned to the normal level in Groups 4 and 5. The intraperitoneally injected tramadol （10 and 20 mg/kg） produced a potent and dose-dependent antinocicptive effect on the lesioned paw. The antinocicptive effects of tramadol were partially an- tagonized by naloxone （200 μg/kg）, suggesting an additional non-opioid mechanism. Conclusion： The results suggest that tramadol could be a good choice for the treatment of pain under the conditions that immunosuppression may be particularly contraindicated.     

Carboxymethytl Pachymaram Up-Regulates Dendritic Cell＇s Function in Hepatitis B Virus Transgenic Mice

The effect of carboxymethytl pachymaram (CMP) on the function of dendritic cells(DCs) derived from spleens of hepatitis B virus transgenic mice are studied in vitro. The phenotypes of DCs are tested by flow cytometry (FCM), cytokines measured by ELISA. The expression of DCs’ phenotypes in HBV transgenic mice are low (CD80⁺CD11c⁺:59.12±11.53 vs 9.60±4.53, p<0.01; CD80⁺ MHC-II⁺: 44.86±12.31 vs 9.80±5.72, p<0.01, normal mice vs HBV transgenic mice), the ability of DCs stimulating T lymphocytes proliferation decreases (0.37±0.11 vs 0.20±0.11, p<0.05, normal mice vs HBV transgenic mice), levels of IL-12 and IFN-γ decrease whereas the level of IL-10 increases; CMP can enhance DCs’ ability of stimulating T lymphocytes proliferation, facilitate the secretion of IL-12 and IFN-γ, inhibit the secretion of IL-10, thus up regulates DCs function. The results show a good prospective use of CMP on the treatment of chronic hepatitis B. Biography: HOU Anji (1963–), male, Ph.D. candidate, Associate professor, research direction: clinical virology.     

一种中药复方对小鼠脾脏淋巴细胞PD-1/PD-L1表达的影响

为了探讨中药复方对小鼠脾脏淋巴细胞PD-1/PD-L1的表达情况,以及CD3~+CD4~+和CD3~+CD8~+T淋巴细胞比例的影响,采用传统熬制中药的方法得到中药复方提取液,按低、中、高3个浓度对小鼠灌胃给药(1次/d),2周后,采用脑脊髓脱臼法处死小鼠,利用Real-time PCR和流式细胞仪检测脾脏PD-1/PD-L1的表达情况以及淋巴细胞亚型的变化。结果表明,随着中药提取液浓度的增加,脾脏淋巴细胞PD-1的表达显著降低,而PD-L1的表达无显著变化;CD3~+CD4~+型和CD3~+CD8~+型T淋巴细胞的比例均显著增加。所研制的中药复方能抑制淋巴细胞PD-1的表达,激活免疫辅助细胞和效应细胞,有望应用于抗肿瘤的免疫治疗。     

Effect of MTEC1 on the functional expression of TCRaβ+ 3G11− 6C10− CD4+ CD8− thymocyte subset

A murine CD4⁺ thymocyte subset with phenotype of TCRαβ⁺ 3G11⁻ 6C10⁻ CD4⁺ CD8⁻ CD69^+/- HSA^med/lo contains the cells in relatively functional matured status. The functional property of the cells in this subset is characterized by the unique pattern of cytokine production at transitional stage from Th0 to Th2 type with the latter being the dominant type. After being co-cultured with murine thymic medullary epithelial cell line (MTEC1) cells, a murine thymic medullary type epithelial cell line, the TCRαβ⁺ 3G11⁻ 6C10⁻ CD4⁺ CD8⁻ CD69^+/- HSA^med/lo thymocytes, has exhibited significantly higher levels of proliferation capability and IL-6 production, whereas the production of IL-4 and IL-10 is suppressed after co-culturing with MTECl. By contrast, MTECl could not induce thymocytes to secrete Thl type of cytokines. The results suggest that MTECl can regulate functional status of this thymocyte subset and induce them to develop into a specialized Th2 subset.     

模拟微重力大鼠T淋巴细胞发育异常的初步研究

对模拟空间微重力条件下大鼠T细胞发育受阻的神经免疫调节机理进行初步探究.采用流式细胞检测技术测定模拟微重力大鼠胸腺中不同发育阶段T淋巴细胞的数量变化,用ELISA法测定胸腺组织中去甲肾上腺素(NE)和内源性糖皮质激素(GC)的含量.结果表明模拟微重力模型大鼠出现明显胸腺萎缩(p<0.05),CD3⁺CD4⁺CD8^-和CD3⁺CD4^-CD8⁺T细胞数量明显减少(p<0.05);模拟微重力大鼠胸腺中NE和GC含量有升高的趋势.模拟微重力大鼠胸腺中发生了从双阳性T细胞向单阳性T细胞的发育阻滞,这种异常可能与神经内分泌免疫调控相关.     

Osseointegration of hollow porous titanium prostheses loaded with cancellous bone matrix in rabbits

This study aimed to observe the osseointegration of hollow porous titanium prostheses (HPTP) loaded with cancellous bone matrix (CBM) in rabbits using histological and biomechanical perspectives.Experimental samples were implanted into the lateral femoral condyles of 66 New Zealand rabbits,allocated into the following groups:non-porous prosthesis group (Group A,n=22);HPTP group (Group B,n=22);HPTP+CBM group (Group C,n=22).The rabbits were sacrificed at 3,8 and 12 weeks,postoperatively.X-ray analyses,microscopy techniques and morphological measurement software and mechanical tests were used for evaluation.At each time point,the tissues surrounding the implants were similar in all of the groups,with bony in-growth into the 2-mm round holes observed for the defects in Groups B and C.However,the internal bone formation was significantly better in Group C than in Group B at different time points (P<0.01).Biomechanically,the pull-out forces were significantly greater in Groups B and C than in Group A (P<0.01),with no difference between Groups B and C (P>0.05).These results suggest that bone can grow into the cavities of HPTP to achieve more stable locking fixation,and those osteogenic materials,such as CBM,can enhance osteogenesis to achieve better osseointegration between the implant and the host bone.     

珍珠贝肉提取液抗衰作用的动物实验

在实验室检测马氏珍珠贝肉提取液对老龄小白鼠分辩学习及记忆保持能力的影响 ,并于行为实验结束后测定海马突触体内游离钙水平变化。小白鼠 1 6～ 1 7月龄 ,体重 (65±4) g,雌雄兼用 ,共 30只 ,分为天然提取液组 1 0只 ,强化提取液组 (天然提取液 +牛磺酸 ) 1 0只 ,对照组 (饮用普通清水 ) 1 0只。平均每只小鼠每天服用量为 5 ml,共服用 40 d。珍珠贝肉天然提取液和牛磺酸强化提取液与对照组相比 ,都明显增强老龄鼠在 Y-迷宫中的分辩学习能力和学习速度 ,减少训练次数 (P <0 .0 5) ;对记忆保持率的提高具有促进作用 ,尤其是牛磺酸强化提取液使记忆保持率大幅度提高 (P <0 .0 1 )。天然提取液与强化提取液都一致降低鼠脑的海马突触体游离钙离子水平 ,提示这种提取液对衰老脑细胞的“钙超载”有拮抗效应     

CD8<Superscript>+</Superscript> T cell response mediates the therapeutic effects of oncolytic adenovirus in an immunocompetent mouse model

The role of anti-tumor immune responses in oncolytic adenoviral therapy has not been well studied due to lack of efficacious tu- mor model in immunocompetent mice.Here,we evaluated the contributions of immune components to the therapeutic effects of oncolytic adenoviruse in an immunocompetent murine tumor model permissive for infection and replication of adenovirus.We found that CD8+T cells were critical mediator for antitumor efficacy by oncolytic adenovirus.Intratumoral viral therapy induced intensive infiltration of CD8+T cells in tumor,increased tumor-specific IFN-?(interferon-?)production and CTL(cytotoxic T lymphocyte)activity of lymphocytes,and generated a long-term tumor-specific immune memory.Boosting CD8+T cell responses by agonistic anti-4-1BB(cluster differentiation 137,CD137)antibody showed synergistic anticancer effects with oncolytic viro- therapy.Our results provide insight into antitumor mechanisms of oncolytic adenovirus in addition to their direct oncolytic effect.     

Prophylaxis of tumor through oral administration of IL-12 GM-CSF gene carried by live attenuated salmonella

A live attenuated AraA^- autotrophic mutant ofSalmonella typhimurium (SL3261) was used as carrier for eukaryotic expression vectors EGFPN1, pCMVmIL-12, pCMVhIL-12, pCMVmGM-CSF and pCMVhGM-CSF and was administered orally to BALB/c and C57BL/6 mice. After 6 weeks, these mice were challenged with 4T1 and Lewis tumor cells respectively. GFP expression and gene integration could be detected in mice’s livers, spleens, intestines, kidneys and tumors. The serum level of cytokines increased significantly in treated mice, so did the ratio of CD ₈ ⁺ /CD ₄ ⁺ , which resulted in the tumor regression and prolongation of the survival time of those mice. These researches laid an experimental foundation for the tumor gene therapy using live attenuated salmonella.     

Phenotypic analysis of the medullary-type CD4−CD8+ thymocytes in mice

Phenotypic analysis of the medullary-type CD4 CD8⁺ (CD8SP) thymocytes has revealed phenotypic heterogeneity within this cell population. The phenotype of mature peripheral CDS⁺T cells is TCRαβ⁺CD3⁺Qa-2⁺HSA 3G11⁻6C10, whereas in the medullary-type CD8SP thymocytes, 20% are Qa-2⁺; 33%, HAS; 30%, 3G11; and 70% are 6C10. The disparate expression patterns of these four cell surface markers suggest that medullary-type CD8SP thymocytes may undergo phenotypic maturation process. According to the distribution of these four cell surface markers, six subgroups of CD8SP thymocytes have been identified. The precursor-progeny relationship along with developmental pathway is postulated as follows: 6C10⁺HSA⁺3G11 Qa-2→ 6C10⁺HSA⁺ 3G11⁺Qa-2 → 6C10 HSA⁺3G11⁺Qa-2 → 6C10⁻HSA⁻3G11⁺Qa-2 → 6C10⁻HSA⁻3G11 Qa-2 → 6C10⁻HA S 3G11 Qa-2⁺, the cells in the last subgroup exit the thymus and home into periphery.     

STAT1 signaling is required for optimal Th1 cell differentiation in mice

Although IFN-γ alone does not prime type I T helper cell (Th1) differentiation, the loss of IFN-γ signaling leads to impaired Th1 phenotype: IFN-γ receptor-deficient (Ifngr-/-) Th1 cells fail to permanently repress IL-4 expression. They can differentiate into IL-4-producing cells under Th2-inducing conditions. These observations suggest that IFN-γ signaling plays a critical role in si- lencing Il4 gene in Th1 cells and stabilizing Th1 phenotype. IFN-γ signaling has been further shown to inhibit IL-4 express...     

Total thyroidectomy is safer with identification of recurrent laryngeal nerve

Objective： To investigate the effect of recurrent laryngeal nerve （RLN） identification on the complications after total thyroidectomy and lobectomy. Methods： Total 134 consecutive patients undergoing total thyroidectomy or thyroid lobectomy from January 2003 to November 2004 were investigated retrospectively. Patients were divided into two groups： RLN identified （Group A） or not （Group B）. The two groups were compared for RLN injury and hypocalcaemia. Results： The numbers of patients and nerves at risk were 71 and 129 in Group A, and 63 and 121 in Group B, respectively, RLN injury in Group A （0） was significantly lower than that in Group B （5 [7.9%]） patients, 7 [5.8%] nerves） for the numbers of patients （P=0.016） and nerves at risk （P=0.006）. Temporary hypocalcaemia was significantly higher in Group A than in Group B （14 [24.1%] vs 6 [ 10.3%], P=0.049）, Permanent complications in Group B were significantly higher than those in Group A （13 [20.6%] vs 4 [5.6%], P=0.009）. Conclusion： RLN injury was prevented and permanent complications were decreased by identifying the whole course and branches of the recurrent laryngeal nerve during total thyroidectomy,     

Relationships among magnetic resonance imaging, histological findings, and IGF-I in steroid-induced osteonecrosis of the femoral head in rabbits

Objective: To study the relationships among magnetic resonance imaging (MRI), histological findings, and insulin-like growth factor-I (IGF-I) in steroid-induced osteonecrosis of the femoral head in rabbits. Methods: Thirty rabbits were randomly divided into experimental Group A (n=15) and control Group B (n=15). The 7.5 mg/kg (2 ml) of dexamethasone (DEX) and physiological saline (2 ml) were injected into the right gluteus medius muscle twice at one-week intervals in animals of Groups A and B, respectively. At 4, 8 and 16 weeks after obtaining an MRI, the rabbits were sacrificed and the femoral head from one side was removed for histological study of lacunae empty of osteocytes, subchondral vessels, and size of fat cells under microscopy, and the femoral head from the other side was removed for enzyme-linked immunoadsorbent assay (ELISA) for IGF-I. Results: At 4, 8 and 16 weeks after treatment, no necrotic lesions were detected in Group B, while they were detected in Group A. Light microscopy revealed that the fat cells of the marrow cavity were enlarged, subchondral vessels were evidently decreased, and empty bone lacunae were clearly increased. The IGF-I levels in Group A were significantly higher than those in Group B. At 8 weeks after the DEX injection, the MRI of all 20 femora showed an inhomogeneous, low signal intensity area in the femoral head, and at 16 weeks, the findings of all 10 femora showed a specific “line-like sign”. The MRI findings of all femora in Group B were normal. Conclusion: MRI is a highly sensitive means of diagnosing early experimental osteonecrosis of the femoral head. However, the abnormal marrow tissues appeared later than 4 weeks when the expression of IGF-I increased. This reparative factor has an early and important role in response to steroid-induced osteonecrosis of the femoral head, and provides a theoretical foundation for understanding the pathology and designing new therapies.     

凝胶化改性壳聚糖膜预防感染性腹膜粘连的对比研究

目的对比研究凝胶化改性壳聚糖膜和透明质酸钠对感染性腹膜粘连的预防作用。方法SD大鼠80只,随机分成四组:假手术对照组(A组),感染对照组(B组),凝胶化改性壳聚糖膜组(C组),透明质酸钠组(D组)。利用大鼠蚓突盲端制造感染性腹膜粘连模型,然后每组分别用相应的方法处理污染面,处理后2、4周打开腹腔,以Bha-tia分级法评定蚓突盲端的粘连程度,并对盲端组织行羟脯胺酸(OHP)水平测定和病理学检查。结果术后2周和4周,C组的粘连程度显著轻于B组(P=0.000),OHP水平显著低于B组(P=0.000);D组的粘连程度、OHP水平和B组无显著性差异(P>0.05)。病理学检查:B组术后2周局部组织以急性化脓性炎症为主,4周时以肉芽组织增生和纤维组织形成的慢性炎症反应为主;C、D组的病理改变类型同B组相同,但C组的上述改变较B组明显减轻,而D组和B组间则无明显差异。结论凝胶化改性壳聚糖膜具有明显的预防感染性腹膜粘连的作用,而透明质酸钠的作用则不明显。     

SLE患者外周血淋巴细胞表达BLyS和CD86的变化

采用FACS研究了 2 2例SLE患者外周血淋巴细胞表达BLyS和CD86的变化。结果表明 ,SLE患者外周血BLyS 淋巴细胞和CD19 CD86 淋巴细胞显著增加 ,但二者之间无显著相关。     

谷氨酰胺对大强度跑台训练大鼠免疫功能影响的实验研究

目的:探讨补充谷氨酰胺对大强度跑台训练大鼠免疫功能的影响。方法:适应性训练后,30只6周龄健康雄性SD大鼠,随机分为安静组(C,6只),训练组(T,12只)和服药训练组(TG,12只)。进行6周大强度跑台训练,跑台坡度5°,前2周每周训练6d,每次训练25min,速度依次为20 m/min和24 m/min,后4周每天训练,速度和时间不变,分别为32 m/min和35min。训练期间每日给药前称体重,TG组大鼠按O.3g/kg.d剂量灌胃给药,C、T组喂以相同体重比例的自来水。最后一次训练12h后进行大鼠脾脏T淋巴细胞亚群CD4+、CD8+和脾细胞IL-2表达测定。结果:T组与C组比较,CD8+含量显著升高,CD4+/CD8+比值及IL-2表达显著下降;TG组与C组比较,CD8+含量显著下降,CD4+/CD8+及IL-2表达升高显著。结论:本该实验的大强度跑台训练造成大鼠免疫功能显著下降,而谷氨酰胺补充可明显改善大强度跑台训练造造成的大鼠免疫抑制,增强大鼠免疫功能。     

Transient expression of IL-2 receptor precedes the differentiation of immature thymocytes

The growth of mature T lymphocytes after activation by antigen is regulated by the binding and endocytosis of interleukin-2 (IL-2). In the thymus, approximately 50% of adult thymocytes that carry neither the CD4 nor the CD8 antigen and day 14-15 fetal CD4-8- thymocytes express receptors for IL-2(IL-2R). The CD4-8- (double-negative) subpopulation of thymocytes contains the precursors of cells that can differentiate along an unknown pathway into thymocytes bearing either CD8 or CD4, with the characteristics of mature T lymphocytes. The basis for IL-2R expression by double-negative thymocytes is unclear as they appear to lack a functional T-cell receptor/CD3 complex through which activation of peripheral T cells is mediated. The argument for a role for IL-2 in thymocyte differentiation has also been complicated by conflicting reports on the inability or capability of double-negative thymocytes to respond to IL-2 in vitro. At present, both the nature of the stimuli within the thymic micro-environment which induce IL-2R expression and its relevance to thymocyte differentiation are not known. We show here that the IL-2R-bearing subset has a greater potential to differentiate into phenotypically mature T lymphocytes than do IL-2R-negative thymocytes. In addition, progeny of IL-2R-negative donor cells transiently express IL-2R in the thymuses of adoptive hosts before generating CD8 and/or CD4-positive thymocytes. These results identify the IL-2R-positive cells as a more differentiated double-negative thymocyte subset on the pathway to mature T lymphocytes.