PC和KC在贮脂细胞激活过程中的协同作用

近年来，对人和实验性肝纤维化发病机理的研究焦点主要集中于贮脂细胞（ＦＳＣ）的病理学改变，而有关正常或不完全损伤肝实质细胞（ＰＣ）在ＦＳＣ激活过程中的作用少见报道。本文对正常和ＣＣｌ４损伤大鼠ＰＣ进行分离培养，观察ＰＣ和库普弗细胞（ＫＣ）在ＦＳＣ激活过程中的协同作用。采用ＰＣ、ＫＣ和ＦＳＣ分离培养，用流式细胞仪测定ＤＮＡ含量，免疫组化和图像分析对ＦＳＣ中Ⅱ、Ⅲ、Ⅳ型胶原和ＦＮ进行定量分析，液闪法检测ＦＳＣ中３Ｈ－脯氨酸掺入量。来自正常或ＣＣｌ４损伤大鼠ＰＣ条件培养基（ＰＣｎｃｍ、ＰＣｔｃｍ）对原代培养ＦＳＣ的增殖具有较强的刺激效应，ＦＳＣ内３Ｈ－脯氨酸掺入量呈现时间和剂量相关性地升高，ＤＮＡ含量在时间和剂量上也呈正相关的增加。单独用ＰＣｎｃｍ或ＰＣｎｃｍ＋ＰＣｔｃｍ合用时，ＦＳＣ表现出明显地转化特征，而ＦＳＣ内Ⅰ、Ⅲ、Ⅳ型胶原和纤维粘连蛋白（ＦＮ）均显著升高。ＰＣ和ＫＣ对ＦＳＣ的激活具有协同效应，并呈现时空相关性，对ＦＳＣ内ＥＣＭ合成的协同效应与增加细胞总量及增加单个ＦＳＣ合成ＥＣＭ的能力有关。     

不同类型胶质瘤移动性的比较及其与细胞外基质成分关系的研究

用３株不同类型的恶性胶质瘤进行细胞体外移动性及其与细胞外基质成分关系的比较研究。结果表明，髓母细胞瘤移动性最强，多形性胶质母细胞瘤次之，ＳＷＯ－３８胶质瘤最弱。３株胶质瘤体外移动性受Ⅰ型胶原，Ⅳ型胶原，层粘连蛋白（ｌａｍｉｎｉｎ，ＬＮ）和纤维结合蛋白（ｆｉｂｒｏｎｅｃｔｉｎ，ＦＮ）等细胞外基质成分的影响基本上是一致的。故不能以此解释其移动性差异的原因。Ⅳ型胶原蛋白和ＬＮ能促进瘤细胞在体外的移动，但Ⅳ型胶原和ＬＮ两者之间不表现出叠加作用，也无拮抗作用。Ⅰ型胶原网能明显地阻遏肿瘤细胞的体外移动性。ＦＮ对这３株胶质瘤的移动性影响不大，但对非神经系统肿瘤（ＧＬＣ－８２肺腺癌）却有较明显的趋触作用。     

血小板衍生性生长因子对体外培养人肾小球系膜细胞生长的影响

目的观察血小板衍生性生长因子（ＰＤＧＦ）对体外培养的人肾小球系膜细胞（ＭｓＣ）生长、基质合成和分泌以及ｃ┐ｍｙｃ癌基因表达的影响。方法体外培养的ＭｓＣ培养液中掺入５┐溴脱氧尿嘧啶（ＢｒｄＵ）,采用ＢｒｄＵ单克隆抗体免疫组化法检测ＭｓＣ的增殖情况;应用［３Ｈ］脯氨酸掺入酶消化法测定ＭｓＣ细胞内、外胶原蛋白总量;采用Ｎｏｒｔｈｅｒｎ印迹法检测纤维连接蛋白（ＦＮ）、Ⅳ型胶原、核癌基因ｃ┐ｍｙｃｍＲＮＡ表达结果。结果（１）ＢｒｄＵ掺入法的阳性细胞标记指数在对照组为１９．５％,ＰＤＧＦ组为３４．５％（Ｐ＜０．０１）;（２）ＭｓＣ经ＰＤＧＦ作用后,细胞内、外胶原蛋白量分别为２．６９±０．６０％和３．８７±０．６５％,较正常对照组１．２５±０．５０％和１．６１±０．５１％明显增加（Ｐ＜０．０１）;（３））Ｎｏｒｔｈｅｒｎ印迹法显示ＰＤＧＦ组细胞的ＦＮ、Ⅳ型胶原及ｃ┐ｍｙｃｍＲＮＡ的表达明显高于正常组。结论ＰＤＧＦ不仅可刺激肾小球ＭｓＣ的增殖和胶原蛋白合成,而且可从基因转录水平增加ＦＮ、Ⅳ型胶原及ｃ┐ｍｙｃ癌基因的表达。由此推断ＰＤＧＦ在肾小球疾病的发生、发展中可能起着重要作用     

钙调素拮抗剂对HeLa细胞S期进程的影响

目的　探讨钙调素拮抗剂三氟拉嗪（ＴＦＰ）对ＨｅＬａ细胞Ｓ期进程的影响及其分子机理。　方法　通过ＴｄＲ双阻断法获得同步化的Ｓ期ＨｅＬａ细胞,以３Ｈ－ＴｄＲ掺入法和Ｗｅｓｔｅｒｎ 技术分别观察了ＴＦＰ对ＨｅＬａ 细胞Ｓ期进程和胸苷激酶（ｔｈｙｍ ｉｄｉｎｅ ｋｉｎａｓｅ,ＴＫ）活性及细胞周期引擎分子ＣｙｃｌｉｎＡ、Ｃｄｋ２ 和细胞周期调节蛋白ｐ８０ｃｄｃ２５Ｂ、ＰＣＮＡ、ｐ２１ 蛋白表达水平的影响。　结果　ＴＦＰ（２０μｍ ｏｌ／Ｌ）能使３ Ｈ－ＴｄＲ的掺入峰值后移,并抑制了Ｃｙ－ｃｌｉｎＡ、ＰＣＮＡ、ｐ８０ｃｄｃ２５Ｂ的表达和提高了ｐ２１ 蛋白的水平,但对Ｃｄｋ２的表达几乎无影响。　结论　ＣａＭ 除了能通过影响ＰＣＮＡ的水平直接作用于ＤＮＡ 合成,同时又可通过作用于ＣｙｃｌｉｎＡ、ｐ８０ｃｄｃ２５Ｂ、ｐ２１ 等周期引擎分子和调控因子水平正调ＨｅＬａ 细胞Ｓ期进程。     

右室快速起搏犬心室扩张及其逆转时心肌胶原和心功能的变化

目的和方法：观察犬心室扩张和逆转时心肌胶原含量、Ⅰ／Ⅲ比值变化及其对心功能的影响。１７只家犬，随机分为①持续起搏（ＰＣ组，ｎ＝６）组，以２５０次／ｍｉｎ频率起搏４周。②恢复（ＲＣ组，ｎ＝６）组，持续右室快速起搏４周停止起搏，恢复４周。③假手术（ＳＯ组，ｎ＝５）组。ＰＣ、ＲＣ组分别于起搏和恢复４周后，ＳＯ组于假手术１周后进行血流动力学、超声心动图以及心肌胶原检测；以羟脯氨酸法测定心肌胶原含量；十二烷基硫酸钠一聚丙烯酰胺凝胶电泳（ＳＤＳ－ＰＡＧＥ）法测定心肌胶原Ⅰ／Ⅲ型比值。结果：与ＳＯ组比，ＰＣ组平均肺毛细血管楔压（ＭＰＣＷＰ）、平均右房压（ＭＲＡＰ）、平均右室压（ＭＲＶＰ）显著增高，心排量（ＣＯ）、平均动脉压（ＭＡＰ）、左室射血分数（ＥＦ）明显下降，心肌胶原含量增加，Ⅰ／Ⅲ型胶原比值显著降低（Ｐ＜００５），而ＲＣ组血流动力学改善，心肌间质胶原及Ⅰ／Ⅲ型胶原比值降低，停止起搏扩张心室逆转后心功能改善，胶原含量和成分也随之恢复     

人血小板生成素cDNA转染COS-7细胞及表达产物对小鼠骨髓巨核细胞的作用

将克隆全长１．１ｋｂ血小板生成素（ＴＰＯ）ｃＤＮＡ构建重组ＰＣＩｎｅｏＴＰＯ表达载体，利用ＬｉｐｏｆｅｃｔｉｎＴＭ介导转染ＣＯＳ－７细胞中，对阳性克隆ＣＯＳ－７细胞的ＤＮＡ、ｍＲＮＡ和培养上清分别进行ＰＣＲ扩增Ｓｏｕｔｈｅｒｎｂｌｏｔ、斑点杂交和ＴＰＯ夹心ＥＬＩＳＡ检测，观察表达产物对小鼠骨髓巨核细胞的作用。结果表明：全长１．１ｋｂＴＰＯｃＤＮＡ转染ＣＯＳ－７细胞获得表达，最高表达量可达４８．２８Ｕ／ｍｌ，其产物使骨髓巨核细胞集落形成单位（ＣＦＵ－ＭＫ）集落数增加４倍，巨核细胞增大至４２．１０±６．７０μｍ（Ｐ＜０．０１）；动态观察骨髓ＣＦＵ－ＭＫ集落数于实验第８天达最高峰，约持续２ｄ，第１０天后开始下降。提示ＴＰＯｃＤＮＡ转染ＣＯＳ－７细胞表达产物对骨髓巨核细胞生成有刺激活性     

地高辛标记的cDNA探针检测GM-CSFmRNA表达

采用随机引物法对人ＧＭ－ＣＳＦｃＤＮＡ 探针进行地高辛标记,核酸分子原位杂交技术对人胸腺细胞、骨髓基质细胞、白血病细胞株（ＨＬ－６０,Ｋ５６２）、脐血细胞和胎肝组织的ＧＭ－ＣＳＦｍ ＲＮＡ的表达水平进行检测。结果表明：地高辛标记的ＧＭ－ＣＳＦｃＤＮＡ 探针使用简便、灵敏度高、特异性强、杂交结果直观,探针可较长时间保存和无放射性污染等优点。     

用噬菌体抗体库分离NK细胞抑制受体特异性的ScFv片段

目的探讨从噬菌体抗体库分离杀伤细胞抑制受体（ＫＩＲｓ）特异性ＳｃＦｖ（ｓｉｎｇｌｅｃｈａｉｎＦｖ）的可行性及其特征。方法所用ＫＩＲ为ＮＫＡＴ２,其可溶性形式为ＮＫＡＴ２－ＩｇＧ１。噬菌体抗体库为Ｎｉｓｓｉｍ等报道的半合成抗体库。抗体库经包被免疫管的ＮＫＡＴ２－ＩｇＧ１５次筛选后,可溶性ＳｃＦｖ由ＩＰＴＧ诱导细菌而获得,筛选效果由测定噬菌体的菌落形成单位及ＤＮＡ酶切图谱分析。ＥＬＩＳＡ、聚丙烯酰胺凝胶电泳及免疫印迹分析ＳｃＦｖ特异性及免疫学特征,并进行ＤＮＡ的序列测定。结果５次筛选后,噬菌体得到２００倍的富集,酶切图谱也显示某种构型噬菌体的富集。４０个克隆的细菌上清经测试,３７个显示与ＮＫＡＴ２有较强的反应。ＳｃＦｖ经聚丙烯酰胺凝胶电泳及免疫印迹显示相对分子质量为３０×１０３,其ＤＮＡ序列完全相同,重链ＣＤＲ３区编码氨基酸为ＥＳＮＬＶＴＣ,重链其它部分与ＤＰ３５一致。结论研究初步结果表明,用噬菌体抗体库可以快速、简便地产生用于ＫＩＲ研究的较高特异性的试剂。     

用于上肢功能神经刺激的抓握合成Ⅰ:用于合成刺激图的自动方法

用于上肢功能神经刺激的抓握合成Ⅰ：用于合成刺激图的自动方法［英］／Ｋｉｌ－ｇｏｒｅＫＬ…／／ＭｅｄＢｌｏｌＥｎｇＣｏｍＰｕｔ．一１９９３，３１（６）．一６０７功能神经刺激（ＦＮＳ—ＦｕｎｃｔｉｏｎａｌＮｅｕｒｏｍｕｓｃｕｌａｒＳｔｉｍｕｌａｔｉｏｎ）...     

抗Ⅲ型前胶原肽单克隆抗体的制备及初步应用

制备出两株分泌抗ＰⅢＰ（Ⅲ型前胶原肽）单克隆抗体的杂交瘤细胞株。竞争抑制ＥＬＩＳＡ试验表明：两株单抗与ＰＣＩ（Ⅰ型前胶原）及ＣⅢ（Ⅲ型胶原）无交叉反应。Ｄｏｔ－ＥＬＩＳＡ示两株单抗钧针对ＰⅢＰ构象决定簇。应用所制备的单抗对人肝细胞癌组织及大鼠、小鼠成纤维母细胞进行免疫定位。结果显示两种成纤维母细胞呈明显膜阳性；肝癌细胞浆内呈粗颗粒状染色，提示肝癌细胞具有合成Ⅲ型前胶原的能力。     

Effect of angiotensin-(1-7) and angiotensin II on the proliferation and activation of human endometrial stromal cells in vitro

Recent studies have shown that angiotensin II (Ang II) or angiotensin-(1-7) [Ang-(1-7)] has effect on the proliferation and activation of a variety of cells, however, the exact mechanisms that the role of Ang II or Ang-(1-7) in human endometrial stromal cell (ESCs) remains elusive. Here we demonstrated that Ang II could promote proliferation and activation of ESCs, up-regulated the expression of a-SMA, TGF-β1 and IGF-I, increased the secretion of extracellular matrix [Type I collagen (Col I) and fibronectin (FN)] of ESCs; Ang-(1-7) could inhibit Ang II induced the proliferation and activation of ESCs, down-regulated the expression of a-SMA, TGF-β1 and IGF-I, decreased the secretion of extracellular matrix (Col I and FN) of ESCs. These findings suggest that Ang-(1-7) can inhibits Ang II induced the proliferation of ESCs, Ang-(1-7) can inhibits the Ang II induced activation of ESCs and decreases secretion of Col I and FN by suppressing TGF-β1 and IGF-I expression.     

Modulation of keratinocyte motility. Correlation with production of extracellular matrix molecules in response to growth promoting and antiproliferative factors.

Normal human epidermal keratinocytes (KC) grown under conditions that maintain the undifferentiated state are highly motile. Migration of these cells as measured in two different assays (migration out of an agarose drop explant, and into micropore filters in a modified Boyden chamber), is stimulated by fibronectin (FN) and to a lesser extent by thrombospondin (TSP). In contrast, laminin (LN) inhibits KC migration. Cultivation of the cells for 1 day under conditions that induce differentiation (ie, in the presence of 1.4 mM Ca2+) suppresses KC motility. A number of soluble growth modulating polypeptide factors also influence KC migration. Transforming growth factor-beta (TGF-beta) and epidermal growth factor (EGF) stimulate KC motility. These factors simultaneously induce KC production of FN and a significant portion of the stimulated motility can be inhibited with antibodies to FN. EGF and somatomedin-C (SM-C), but not TGF-beta, also stimulate TSP production while EGF and SM-C (but not TGF-beta) induce KC proliferation. In contrast to these factors, interferon-gamma (INF-gamma) inhibits KC production of both FN and TSP and concomitantly inhibits both motility and proliferation. These data suggest that KC properties essential for normal wound healing (ie, motility and proliferation) are regulated by both extracellular matrix molecules and soluble peptide factors. Finally, these effects of various growth promoting and antiproliferative factors on KCs may, in part, be mediated through alteration in the endogenous production of extracellular matrix molecules by KCs.     

Proliferation of fat-storing cells is stimulated by secretions of Kupffer cells from normal and injured liver

The influence of Kupffer cells (KC) from normal, D-galactosamine- and thioacetamide-injured liver on the growth of rat liver fat-storing cells (FSC) in culture was studied. The supplementation of FSC incubation medium with normal and galactosamine-KC-conditioned media for 4 days caused 1.5- and 1.9-fold increase (P less than 0.005), respectively, in the DNA contents of FSC on the fifth day of culture. The stimulant effect reached a maximum at a 1:2 dilution of the conditioned medium with FSC incubation medium. The population doubling time of FSC grown in the absence of conditioned medium was calculated to be 41.5 +/- 3 hr; it was greatly shortened by KC-conditioned medium from galactosamine-treated rats (24 +/- 2 hr), by normal KC-conditioned medium (28 +/- 4 hr), and by KC medium from thioacetamide-injured rats (33 +/- 5 hr). Similarly KC-conditioned media of either source stimulated significantly the rate of [3H]thymidine incorporation into DNA of FSC even during short-term exposures of FSC with KC medium. The growth-promoting effect of normal KC-conditioned medium could be enhanced if the Kupffer cells were challenged with zymosan and phorbol esters, respectively, but not with lipopolysaccharide. The light microscopic appearance of FSC grown in the presence of KC-conditioned media was greatly changed: the cultures became more dense; the cells spread and developed long extensions. The size and number of lipid droplets decreased more rapidly than in control cultures maintained without addition of KC-conditioned media. The protein DNA ratio of FSC exposed with KC-conditioned media was reduced due to a strong increase in DNA, which is not followed by a similar increase in cellular protein. If referred to the DNA content of the culture, the incorporation of [3H]valine into the protein of the medium was not changed, that into cellular protein was strongly reduced under the influence of normal KC-conditioned medium. Secretions of Kupffer cells obviously stimulate significantly the proliferation of FSC in culture.     

IGFBP-rP1对肝星状细胞活化及核因子-κB活性的影响

目的：明确胰岛素样生长因子结合蛋白相关蛋白1（IGFBP-rP1）是否具有活化肝星状细胞（HSC）、增加细胞外基质（ECM）合成的作用；并探讨可能的信号转导途径。方法：大鼠肝星状细胞株（HSC-T6）体外培养，分别设立空白对照组（加入等量PBS）和不同浓度的 IGFBP-rP1 处理组，干预因素处理24 h后收集细胞爬片或制备单细胞悬液，采用免疫细胞化学染色观察HSC-T6中α-平滑肌肌动蛋白（α-SMΑ）、I型胶原（collagen I）、纤维连接蛋白（FN）的表达变化；流式细胞仪检测HSC-T6中核因子-κB p65（NF-κB p65）的DNΑ结合活性。结果：免疫细胞化学染色结果发现，IGFBP-rP1各处理组α-SMΑ、collagen I、FN的表达均较空白对照组显著增强，且在一定范围内呈剂量依赖关系；流式细胞仪检测结果显示，IGFBP-rP1各处理组NF-κB p65阳性细胞百分数均较空白对照组明显增高。结论：IGFBP-rP1可以激活HSC，且在一定剂量范围内随着IGFBP-rP1剂量的增加HSC活化的程度逐渐增强；IGFBP-rP1可使ECM的重要组成成分collagen I和FN的合成增加；IGFBP-rP1可增强HSC中NF-κB p65的DNΑ结合活性。     

纤维粘连蛋白对培养的自发性高血压大鼠心肌成纤维细胞增殖及胶原合成的影响

目的:观察纤维粘连蛋白(FN)对培养的SHR和WKY大鼠心肌成纤维细胞(CFb, CFb_SHR, CFb_WKY)增殖及胶原合成的影响。方法:CFb取自12周的SHR和WKY大鼠, 采用组织块贴壁法培养, 以直接细胞计数法和 [³H]-TdR掺入率反映细胞增殖, 以[³H]-脯氨酸([³H]-proline)掺入率反映胶原合成。使用FN(5 μg/cm²)预先处理24孔培养板。 结果: 与0.4% FCS对照组相比, 经72 h孵育FN明显促进CFb_SHR和CFb_WKY细胞数增多, 分别为对照组的163.75%(CFb_SHR)和170.42%(CFb_WKY)。FN促进CFb_SHR和CFb_WKY [³H]-TdR掺入增加。FN促进CFb_SHR和CFb_WKY[³H]-proline掺入增加。 结论:FN促进SHR和WKY大鼠的CFb增殖及胶原合成。     

Effects of ECM Protein Mimetics on Adhesion and Proliferation of Chorion Derived Mesenchymal Stem Cells

Background: We evaluated the effects of fibronectin, collagen, cadherin, and laminin based extracellular matrix (ECM) protein mimetics coated with mussel derived adhesive protein (MAP) on adhesion and proliferation of chorionic mesenchymal stem cells (cMSCs).Methods: Human placental chorionic tissues from term third-trimester pregnancies (n=3) were used. The cMSCs were cultured on rationally designed ECM protein mimetics coated with MAP on plastic surfaces with the addition of reduced fetal bovine serum (0.5%, 1% FBS). Adhesion capabilities were monitored by a real time cell analysis system (RTCA) utilizing an impedance method. Proliferation capabilities were monitored by RTCA and MTS assay.Results: Of the ECM protein mimetics tested, GRGDSP(FN) coated surfaces exhibited the highest adhesion and proliferation capabilities on RTCA at FBS concentration of 0.5% and 1%. When 0.5% FBS was added to ECM protein mimetics during the MTS assay, GRGDSP(FN), REDV(FN), and collagen mimetics, GPKGAAGEPGKP(ColI) showed higher cMSCs proliferation compared with the control. When 1% FBS was added, GRGDSP(FN) and TAIPSCPEGTVPLYS(ColIV) showed significant cMSCs proliferation capacity.Conclusions: Fibronectin mimetics, GRGDSP(FN) amino acid sequence showed the highest adhesion and proliferation capabilities. In addition, results from RTCA assessment of cell viability correlated well with the tetrazolium-based MTS assay.     

An ex vivo model for chondrogenesis and osteogenesis

Loss of bone and cartilage are major healthcare issues. At present, there is a paucity of therapies for effectively repairing these tissues sustainably in the long term. A tissue engineering approach using advanced functional scaffolds may provide a clinically acceptable alternative. In this study, an innovative mineralized alginate/chitosan scaffold was used to provide tailored microenvironments for driving chondrogenesis and osteogenesis from single and mixed populations of human articular chondrocytes and human bone marrow stromal cells. Polysaccharide capsules were prepared with combinations of these cell types with the addition of type I or type II collagen to augment cell-matrix interactions and promote the formation of phenotypically distinct tissues and placed in a rotating (Synthecon) bioreactor or held in static 2D culture conditions for up to 28 days. Significant cell-generated matrix synthesis was observed in human bone marrow bioreactor samples containing type I collagen after 21-28 days, with increased cell proliferation, cell activity and osteocalcin synthesis. The cell-generated matrix was immuno-positive for types I and II collagen, bone sialoprotein and type X collagen, a marker of chondrogenic hypertrophy, demonstrating the formation of a mature chondrogenic phenotype with areas of new osteoid tissue formation. We present a unique approach using alginate/collagen capsules encapsulated in chitosan to promote chondrogenic and osteogenic differentiation and extracellular matrix formation and the potential for tissue-specific differentiation. This has significant implications for skeletal regeneration and application.     

JNK信号通路调控大鼠再生肝8种细胞的增殖和凋亡

目的 从基因转录水平了解JNK信号通路在大鼠再生肝8种细胞中的作用。方法 用密度梯度离心和免疫磁珠等方法分离肝细胞（HC）、胆管上皮细胞（BEC）、卵圆细胞（OC）、肝星形细胞（HSC）、窦内皮细胞（SEC）、库普弗细胞（KC）、陷窝细胞（PC）、树突状细胞（DC）等8种肝脏细胞,用大鼠Genome 230 2.0芯片检测大鼠再生肝8种细胞的基因表达谱,用生物信息学和系统生物学等方法分析基因表达变化预示的JNK信号通路在调控大鼠再生肝8种细胞增殖、凋亡中的作用。用实时荧光定量PCR方法验证了芯片结果的可靠性。结果 JNK信号通路涉及240个基因和42条途径,其中,225个基因与大鼠肝再生相关。基因协同作用分析显示,在大鼠肝再生启动阶段,JNK信号通路启动HC和KC增殖,促进OC凋亡,启动部分PC和SEC增殖和促进部分PC和SEC凋亡;在进展阶段,JNK信号通路促进HC、BEC、KC和DC增殖,促进部分PC增殖、部分PC凋亡。在终止阶段,JNK信号通路促进HC、OC和PC凋亡,促进部分KC增殖、部分KC凋亡。结论 大鼠肝再生中JNK信号通路的42条途径和225个基因参与调控大鼠再生肝8种细胞的增殖和凋亡。