Growth and differentiation of adult rat hepatocytes regulated by the interaction between parenchymal and non-parenchymal liver cells

We have devised a medium which supports the continuous growth of hepatocytes without losing their replicative potential and differentiation capacity for a longer period. The medium HCGM, contains four key substances in addition to foetal bovine serum. They are epidermal growth factor, nicotinamide, ascorbic acid 2-phosphate and dimethylsulphoxide. When a non-parenchymal cell fraction containing small hepatocytes and non-parenchymal cells was cultured in HCGM, small hepatocytes grew clonally and differentiated into cells expressing either mature hepatocyte marker proteins or biliary cell marker proteins. Thus, for the first time, we showed the presence of a small compartment of bipotent and highly replicative clonogenic hepatocytes in the rat adult liver. HCGM also supported the growth of stellate cells (Ito cells) which were in the original preparation, suggesting the important role of stellate cells for the successful cultivation of hepatocytes. Together, these results suggest that a microenvironment is produced as a result of cooperative interactions between hepatocytes and stellate cells: one which stimulates the growth and differentiation of clonogenic hepatocytes.     

Primary brain stem lesions caused by closed head injuries

Traumatic lesions of the brain stem are of two types: primary, which are considered to be caused at the moment of impact, and secondary, associated with supratentorial mass lesion. Of the 239 patients with a serious head injury who showed a severe disturbance of consciousness upon admisision and who had CT scan carried out immediately, 21 cases were considered to have a primary brain stem lesion with initial CT scan. A primary brain stem lesion was found in 21 of 239 (8.8%) of patients with serious head injury. Their injuries were caused primarily by traffic accidents. Sixteen of the 21 cases showed not only brain stem lesions but also other brain injuries such as cerebral contusion of the white and gray matter, callosal injury, intraventricular hemorrhage, and subarachnoid hemorrhage, which are considered to be caused by a diffuse shearing injury. Five cases who showed a single injury to the brain stem with no other brain lesions were considered to have a pure brain stem lesion. Primary brain stem lesions were observed on the dorsal side of the midbrain, where they can be differentiated from secondary brain stem lesions. These lesions are considered to result from the shearing mechanism in and around the brain stem very close to the tentorial edge, or to an injury of the lower brain stem by hyperextension of the cervical vertebrae. The prognosis of patients with a primary brain stem lesion was usually unfavorable, except in those with a single brain stem lesion.     

人羊膜间充质干细胞的研究进展

人羊膜间充质干细胞(hAMSC)具有来源丰富、分离简单的优点。hAMSC在组织修复中具有支持造血、再生、免疫调节、抗纤维化等作用。本文对羊膜间充质干细胞在各个系统疾病治疗的研究进行了综述,旨在为羊膜间充质干细胞的应用提供参考。     

淫羊藿苷对人牙髓干细胞神经向分化作用的初步研究

目的 探讨淫羊藿苷（ICA）对人牙髓干细胞（DPSC）神经向分化的作用。方法 分离培养DPSCs后,采用不同浓度ICA(0.01μmol、0.10μmol、1.00μmol、10.00μmol)处理DPSCs。CCK-8法检测ICA对细胞增殖活力的影响,确定其最佳促DPSCs增殖浓度。分别进行细胞形态学观察和Nestin细胞免疫荧光检测。Western blotting检测DPSCs神经向分化相关蛋白Nestin、βⅢ-tubulin及NSE的表达。结果 不同浓度ICA细胞相对活力值比较,差异有统计学意义（P <0.05）,其中以0.10μmol ICA细胞相对活力最高。与对照组比较,ICA组神经球的直径增加（P <0.05）,Nestin荧光强度增强（P <0.05）,Nestin、βⅢ-tubulin、NSE蛋白相对表达量升高（P <0.05）。结论 ICA能够有效促进DPSCs增殖,提高DPSCs神经向分化能力。     

转FL、GM-CSF基因的人骨髓基质细胞促进脐血CD34+细胞体外扩增

研究转FL、GM-CSF基因的基质细胞对脐血CD34+细胞的扩增效应.将转FL、GM-CSF基因的入骨髓基质细胞系与脐血CD34+细胞共培养,观察细胞总数、CD34+细胞数、CFU-GM的变化情况.培养到第4周时,第(4)组(SCF+IL-3+IL-6+GM-CSF+FL)和第(8)组(HFCL/hGM-CSF+HFCL/hFL+SCF+IL-3+IL-6)的细胞总数增加到最大,分别扩增了717±24.47和709±63.63,第1周,第(5)组(HFCL+SCF+IL-3+IL-6)扩增了10.5±2.08倍,较第(8)组减少(P＜0.05).第1周时,CD34+细胞总数第(4)组和第(8)组分别扩增了8.44倍和11.5倍(P＜0.05),CD34+细胞百分率第(7)组(FCL/hFL+SCF+IL-3+II,-6)为50.2%,第(6)组(HFCL/hGM-CSF+SCF+IL-3+IL-6)为28.95%(P＜0.01).第2周,各组CFU-GM增加显著,以第(4)组和第(8)组增加最为明显,以后随扩增时间延长,造血细胞集落数、集落体积逐渐减少.表明转FL、GM-CSF基因的基质细胞,能有效的协同其他细胞因子对脐血CD34+细胞产生明显的扩增作用,能显著改变基质细胞造血功能.     

Characterization of NK cells and extrathymic T cells generated in the liver of irradiated mice with a liver shield

We previously reported that c-kit⁺ stem cells which give rise to extrathymic T cells are present in the liver of adult mice. Further characterization of extrathymic T cells in the liver of adult mice is conducted here. When mice with a liver shield were lethally (9.5 Gy) irradiated, all mice survived. All tested organs showed a distribution pattern of hepatic lymphocytes on day 7. The distribution pattern in the liver was characterized by an abundance of NK (CD3^? IL-2Rβ⁺) and extrathymic T cells (CD3^int IL-2Rβ⁺) before and after irradiation. To determine their function, post-irradiation allogeneic bone marrow transplantation (BMT) was performed in mice with or without a liver shield. Allogeneic BM cells were rejected in mice with a liver shield and specific activation of CD8⁺ CD3^int IL-2Rβ⁺ cells was induced. At that time, potent cytotoxicity of liver mononuclear cells (MNC) against allogeneic thymocytes was induced. Both NK1.1⁺ and NK1.1^? subsets of CD3^int cells expanded in these mice. An in vivo elimination experiment of the subsets indicated that the NK1.1⁺ subset of CD3^int cells (i.e. NK T cells) was much more associated with the rejection of allogeneic BM cells. However, even after the elimination of NK T cells, allogeneic BM cells were rejected. In this case, granulocytes expanded in parallel with NK1.1^? subsets. Granulocytes may also be associated with the rejection of allogeneic BM cells. These results suggest that the liver is an important haematopoietic organ even in adult life.     

Afferent fiber connections from lower brain stem to hypothalamus studied by the horseradish peroxidase method with special reference to noradrenaline innervation

Summary Attempts were made to determine the afferent projections to the anterior hypothalamus including the preoptic area from the lower brain stem by means of the horseradish peroxidase method combined with monoamine oxidase staining to identify noradrenaline (NA) neurons. In addition to this technique, a histofluorescence analysis was performed. NA fibers in the medial part of the anterior hypothalamus were mainly supplied by A1 and A2 NA neuron groups, while the lateral part and periventricular zone received NA terminals from both pontine and medulla oblongata NA neuron groups. Furthermore, the present study indicated that there were direct projections to the anterior hypothalamus from non-noradrenergic neurons in the lower brain stem: nuclei raphe dorsalis, centralis superior, cells in the mesencephalic and pontine central gray matter, nuclei parabrachialis lateralis and medialis, cells around fasciculus longitudinalis medialis.Abbreviations CA Commissura anterior - CO Chiasma opticum - DP Decussatio pyramidum - DPCS Decussatio pedunculorum cerebellarium superiorum - F Columna fornicis - FLM Fasciculus longitudinalis medialis - FMT Fasciculus mamillothalamicus - GCM Griseum centrale mesencephali - GCP Griseum centrale pontis - LL Lemniscus lateralis - LM Lemniscus medialis - PCM Pedunculus cerebellaris medius - PCS Pedunculus cerebellaris superior - TO Tractus opticus - TS Tractus solitarius - TVme Tractus mesencephalicus nervi trigemini - V Ventriculus tertius - VTS Tractus spinalis nervi trigemini - am nucleus ambiguus - B Barrington nucleus - com nucleus commissuralis - cp nucleus caudatus putamen - cs nucleus centralis superior - ct nucleus corporis trapezoidei - cu nucleus cuneatus - dX nucleus dorsalis nervi vagi - Gd nucleus tegmentalis dorsalis (von Gudden) - gr nucleus gracilis - Gv nucleus tegmentalis ventralis (von Gudden) - ha nucleus hypothalamicus anterior - hl nucleus hypothalamicus lateralis - hpe nucleus periventricularis (hypothalami) - hvm nucleus ventromedialis hypothalami - lc nucleus locus coeruleus - oi nucleus olivaris inferior - p nucleus pontis - pa nucleus paraventricularis - pbl nucleus parabrachialis lateralis - pbm nucleus parabrachialis medialis - ph nucleus praepositus hypoglossi - pol nucleus preopticus lateralis - pom nucleus preopticus medialis - pop nucleus preopticus periventricularis - rd nucleus raphe dorsalis - re nucleus reuniens - rl nucleus reticularis lateralis - rm nucleus raphe magnus - ro nucleus raphe obscrus - sc nucleus suprachiasmaticus - so nucleus supraopticus - st nucleus interstitialis striae terminalis - td nucleus tractus diagonalis (Broca) - ts nucleus tractus solitarii - Vme nucleus mesencephalicus nervi trigemini - Vmo nucleus motorius nervi trigemini - Vts nucleus tractus spinalis nervi trigemini - XII nucleus nervi hypoglossi     

兔髓核细胞诱导大鼠骨髓间充质干细胞分化为髓核细胞的研究

目的 研究体外新西兰大白兔髓核细胞(nucleus pulposus cells)与SD大鼠骨髓间充质干细胞(mes-enchymal stem cells,MSCs)共培养时,兔髓核细胞与大鼠MSCs直接和间接接触对MSCs分化为髓核细胞的影响.方法 DAPI (4‘,6-二咪基-2‘-苯吲哚盐酸)标记原代髓核细胞后,分别与第三代MSCs按接触组和非接触组(Transwell培养系统)共培养.每隔24小时应用免疫荧光观察MSCs的形态学变化,并用RT-PCR方法检测Ⅱ型胶原和可凝集蛋白多糖(Aggrecan)的表达.结果 直接接触培养组中可见分化的MSCs形态和功能接近髓核细胞;非直接接触组的MSCs未见变化.结论 髓核细胞与MSCs的直接接触,是诱导MSCs分化为髓核细胞的重要因素.     

鼠巨细胞病毒抑制神经干细胞分化及分化基因表达的体外研究

目的 研究鼠巨细胞病毒(MCMV)感染对体外培养神经干细胞(NSCs)分化及分化基因表达的影响,探讨CMV先天感染致神经损伤的机制.方法 体外分离培养和鉴定BALB/c胎鼠NSCs并检测其分化潜能,用感染复数(MOI)为5、1和0.1的MCMV Smith毒株感染NSCs并进行分化培养,倒置显微镜下观察细胞形态学改变,流式细胞术检测分化细胞比率,免疫荧光法观察NSCs及其分化细胞标记物Nestin、胶质纤维酸性蛋白(GFAP)和神经元特异性烯醇化酶(NSE)表达的变化,采用MCMV早期抗原(EA)示踪感染过程(MOI=1),real-time RT-PCR检测分化早期NSCs Wnt信号途径关键分化基因Wnt-3和Wnt-Ta mRNA水平的动态变化.结果 体外培养的NSCs呈球样生长,神经干细胞特异性标记Nestin表达阳性,并可进一步诱导分化为NF-200阳性的神经元和GFAP阳性的星形胶质细胞;分化培养后,感染组NSCs不能贴壁分化生长并逐渐出现肿胀,细胞Nestin表达下调缓慢并显著高于正常对照组,而GFAP和NSE表达显著低于正常对照组(P<0.05),可检测到MCMV EA的阳性表达;分化培养3-9 d,感染组Nestin阳性细胞比率显著高于正常对照组,GFAP和NSE阳性细胞比率显著低于正常对照组(P<0.05);感染组Wnt-3 mRNA水平在分化培养后第1～2天显著低于正常对照组(P<0.05),感染组Wnto-7a mRNA水平在第0.5～2天明显低于正常对照组(P<0.05);感染组和正常对照组的差异随病毒MOI的增加而更加明显.结论 MCMV感染可明显抑制NSCs向神经元和星形胶质细胞方向分化,导致分化细胞比率减少;下调或干扰NSCs wnt信号途径分化基因wnt-3和Wnt-7a的表达;抑制NSCs分化及其分化基因表达的效应与MOI大小存在一定量效依赖关系;MCMV可能通过抑制NSCs分化基因的表达来抑制其分化,这可能是CMV先天感染致脑发育异常的重要机制之一.