转化生长因子β1对G1期的负性调节

ＴＧＦ是具有生物活性的多肽，它通过下调Ｇ１期细胞周期蛋白Ａ，Ｄ，Ｅ的表达、改变细胞周期蛋白依赖性激酶ＣＤＫ２，ＣＤＫ４和细胞周期蛋白依赖性激酶抑制因子ｐ１６，ｐ１５，ｐ２１，ｐ２７的活性，使细胞停滞在Ｇ１期，ＴＧＦ－β１还可通过抑癌基因产物Ｒｂ非磷酸化状态的聚积和下调Ｇ１早期“演进因子”ｃ－ｍｙｃ的表达阻止Ｇ１期细胞向Ｓ期进展     

抗内毒素单链抗体基因的构建、序列分析及表达

目的：构建抗内毒素（ＬＰＳ） 单链抗体基因, 并尝试其在Ｅ．ｃｏｌｉ 中的表达。方法： 采用ｌｉｎｋｅｒ Ｐｒｉｍ ｅｒ Ｍｉｘ ,按ＶＨｌｉｎｋｅｒＶＬ 的结构将鼠抗ＬＰＳ ｍ Ａｂ Ｃ３Ａ２ 的ＶＨ ,ＶＬ 基因拼接成单链抗体（ＳｃＦｖ） 基因;用ＰＥ３７３Ａ 型全自动ＤＮＡ序列分析仪测定其核苷酸序列。ＰＣＲ 扩增抗ＬＰＳ ＳｃＦｖ 基因并更换两端接头序列后,插入谷胱甘肽巯基转移酶（ＧＳＴ）融合表达载体ｐＧＥＸ４Ｔ１ ;转染Ｅ．ｃｏｌｉ ＪＭ１０９ ,以ＩＰＴＧ 诱导表达,ＳＤＳＰＡＧＥ 分析表达产物。结果：扩增出的ＳｃＦｖ基因长７３５ｂｐ , 序列分析表明,该序列完整、正确;ＳＤＳＰＡＧＥ 显示,转染入重组质粒ｐ４ＴＣ３Ａ２Ｆｖ 的ＪＭ１０９ 菌经诱导后,有相对分子质量（ Ｍｒ） 约为５２ ０００ 的外源蛋白表达。结论：成功地构建了鼠抗ＬＰＳ ＳｃＦｖ 基因,并在Ｅ．ｃｏｌｉ ＪＭ１０９中表达了ＧＳＴＳｃＦｖ 融合蛋白。     

c-erbB-2、p53、bcl-2和nm23-H1在肺癌中的表达

目的：探讨ｃｅｒｂＢ２、ｐ５３、ｂｃｌ２和ｎｍ２３Ｈ１基因在肺癌发生发展过程中的作用。方法：用免疫组化ＡＢＣ法对原发性肺癌组织中４种基因的表达和突变进行检测。结果：５８例肺癌中,３１例（５３４５％）ｐ５３过度表达,１８例（３１０３％）ｂｃｌ２过度表达。ｃｅｒｂＢ２与ｎｍ２３Ｈ１在１０例小细胞肺癌（ＳＣＬＣ）中未见表达。而在４８例非小细胞肺癌（ＮＳＣＬＣｓ）中两者过度表达率均为５０％。ｃｅｒｂＢ２与ｎｍ２３Ｈ１表达呈正相关（Ｐ＜００５）。腺癌ｎｍ２３Ｈ１的表达明显高于鳞癌（Ｐ＜００５）。ｐ５３、ｂｃｌ２蛋白表达在肺癌分化程度中呈负相关（Ｐ＜００５）。ｎｍ２３Ｈ１、ｐ５３和ｂｃｌ２的表达与患者的生存率有关（Ｐ＜００５）。结论：ｃｅｒｂＢ２、ｐ５３、ｂｃｌ２和ｎｍ２３Ｈ１基因蛋白产物的检测对肺癌患者的诊治和预后评估有积极意义。     

以Bacmid-杆状病毒-昆虫细胞系统表达人FGF-9

目的：在Ｂａｃｍｉｄ杆状病毒昆虫细胞系统中表达人ＦＧＦ９ 。方法：采用ＲＴＰＣＲ技术,自新鲜人脑胶质瘤组织获取人ＦＧＦ９ 全编码区ｃＤＮＡ,将其克隆入ｐＣＲＴＭ Ⅱ质粒及ｐＹＥＸ４Ｔ１ 真核表达质粒,经ＤＮＡ自动测序仪进行ＤＮＡ序列测定。将人ＦＧＦ９ ｃＤＮＡ 定向克隆入ｐＦａｓｔＢａｃ 质粒,进一步将其转座入Ｂａｃｍｉｄ 中,在昆虫细胞Ｓｆ９ 中进行表达,采用ＳＤＳＰＡＧＥ对表达产物进行分析。结果：在昆虫细胞表达系统中表达出人ＦＧＦ９ 重组蛋白。结论：人ＦＧＦ９ 在Ｂａｃｍｉｄ杆状病毒昆虫细胞系统中得到了表达。     

树实验肝癌发生过程中IGF-Ⅱ、HBxAg、p21和p53蛋白的表达

目的：探讨ＨＢＶ与ＡＦＢ１协同致肝癌作用机制。方法：用免疫组化观察５３只树鼠句肝及肝癌组织ＩＧＦ－Ⅱ、ＨＢｘＡｇ、ｐ２１及ｐ５３蛋白表达。结果：接受ＨＢＶ和ＡＦＢ１的树鼠句肝癌诱发率和ＩＧＦ－Ⅱ、ＨＢｘＡｇ、ｐ２１检出率分别为５２．９％、８２．４％、５２．９％及２９．４％；只感染ＨＢＶ的分别为１１．１％、２２．２％、１１．１％和１１．１％；仅摄入ＡＦＢ１的分别为１５．８％、２６．３％及１５．８％（ｐ２１）；空白对照无肝癌及上述基因蛋白表达。带瘤肝ＩＧＦ－Ⅱ、ＨＢｘＡｇ及ｐ２１检出率均显著高于无瘤者，而表达这３种基因蛋白的动物肝癌发生率也均明显高于阴性者。突变的ｐ５３蛋白表达仅见于７例（５８．３％）中低分化的肝癌。结论：ＨＢＶ与ＡＦＢ１共同激发这些基因的异常表达可能是其协同致肝癌机制之一。ＩＧＦ－Ⅱ过表达可能作为癌变早期信号应引起重视     

去甲二氢愈创木酸对人恶性胶质瘤细胞系SHG-44生长和分化的影响

目的探讨去甲二氢愈创木酸（ＮＤＧＡ）对胶质瘤生长和分化的作用及可能机理。方法分别将ＮＤＧＡ加入培养基中和进行单细胞胞浆内显微注射ＮＤＧＡ，观察它对人恶性胶质瘤细胞系ＳＨＧ－４４细胞生长、形态、细胞周期和免疫组化特性的影响。结果经ＮＤＧＡ处理的瘤细胞贴壁率和生长速率受抑制，增殖活性降低，细胞周期也有明显改变；细胞异型性变小，胞浆中胶质细丝增多，且其中ＧＦＡＰ标记增加而ｖｉｍｅｎｔｉｎ标记减少；ｐ５３蛋白和碱性成纤维细胞生长因子（ｂＦＧＦ）表达也降低。单细胞胞浆内注射ＮＤＧＡ（约１．５×１０－１１ｇ／细胞）后上述作用更显著，且作用迅速而持久。结论ＮＤＧＡ对恶性胶质瘤细胞具有抑制生长和诱导分化作用；对血管生成因子表达亦有抑制作用。     

抗人肿瘤坏死因子—α单链抗体基因在大肠杆菌中的融合表达

目的：将抗ｈＴＮＦ－α单链抗体基因克隆入融合表达载体ｐＧＥＸ４Ｔ－１中，以期得到ＧＳＴ－ＳｃＦｖ融合表达蛋白。方法：将限制性内切酶酶切拼接法获得的Ｅ６ＳｃＦｖ基因克隆入融合表达载体ｐＧＥＸ４Ｔ－１中，转化大肠杆菌ＤＨ５α，经异丙基－β－Ｄ－硫代半乳糖苷（ＩＰＴＧ）诱导，１２％ＳＤＳ－ＰＡＧＥ检测表达产物，光密度扫描和Ｗｅｓｔｅｒｎ－ｂｌｏｔ验证表达产物。结果：ＳＤＳ－ＰＡＧＥ显示，Ｅ６ＳｃＦｖ表达产物约为５２ｋｕ左右，与预期的结果相符；光密度扫描结果表明，ＧＳＴ－Ｅ６ＳｃＦｖ融合蛋白占菌体总蛋白的４０％；Ｗｅｓｔｅｒｎ－ｂｌｏｔ证实，在相应分子量处，有ＧＳＴ－Ｅ６ＳｃＦｖ融合蛋白的显色印迹；进一步对表达产物的形式分析，ＧＳＴ－Ｅ６ＳｃＦｖ融合蛋白的表达产物为包涵体形式。结论：在大肠杆菌中成功地表达了抗ｈＴＮＦ－α单链抗体基因与谷胱甘肽巯基转移酶（ＧＳＴ）基因的融合蛋白     

单纯疱疹病毒I型胸苷激酶基因在大肠杆菌中的融合表达

用ＥｃｏＲ Ⅰ和Ｈｉｎｄ Ⅲ双酶切已构建的含单纯疱疹病毒Ｉ 型胸苷激酶（ ＨＳＶ１ ＴＫ） 基因的ｐ ＵＣ１８／ＴＫ 质粒，将切出的ＴＫ 基因片段克隆入原核表达载体ｐ ＷＲ４５０１ 中，构建成ＨＳＶ１ ＴＫ 基因重组表达质粒ｐ ＷＲ４５０１／ＴＫ。以ＨＳＶ１ ＴＫ 特异性引物ＴＫ１ Ｆｏｒ 和ＴＫ２ Ｒｅｖ 进行ＰＣＲ 鉴定可扩增出预期的５０３ ｂｐ 的ＴＫ 基因编码区部分序列；ＥｃｏＲ Ⅰ和Ｈｉｎｄ Ⅲ双酶切质粒ｐ ＷＲ４５０１／ ＴＫ， 可切出约１１５０ｂｐ 的ＤＮＡ 片段， 表明重组质粒中已插入ＨＳＶ１ ＴＫ基因片段。用ＩＰＴＧ 诱导ｐ ＷＲ４５０１／ＴＫ／ＪＭ１０９ ，表达出相对分子质量（ Ｍｒ） 约为９７ ０００ 的ＴＫ 和β半乳糖苷酶（ Ｍｒ５５ ０００） 融合蛋白。薄层扫描显示，该融合蛋白含量占菌体蛋白总量的２７ ．４７ ％ 。     

17-β-雌二醇对大鼠垂本前叶细胞转化生长因子基因表达的影响

实验采用无血清原代培养大鼠垂体前叶细胞及原位杂交方法，观察了不同剂量（１０１０、１０８和１０６ｍｏｌ／Ｌ）的１７β雌二醇（ｅｓｔｒａｄｉｏｌ，Ｅ２）对大鼠垂体前叶细胞转化生长因子α（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒα，ＴＧＦα）和转化生长因子β１（ｔｒａｎｓｆｏｒｍｉｎｇｇｒｏｗｔｈｆａｃｔｏｒβ１，ＴＧＦβ１）基因表达的影响。结果表明：１０１０ｍｏｌ／ＬＥ２作用２４ｈ后，对两种生长因子的表达均无显著影响；而１０８ｍｏｌ／Ｌ和１０６ｍｏｌ／ＬＥ２则显著升高ＴＧＦαｍＲＮＡ，分别为对照组的１５倍和１６倍（ｐ＜０００１）；同时明显降低ＴＧＦβ１ｍＲＮＡ，使ＴＧＦβ１ｍＲＮＡ水平分别降低为对照组的７０％和５５％（ｐ＜０００１）。说明一定浓度的Ｅ２对正常大鼠垂体前叶细胞ＴＧＦα和ＴＧＦβ１基因表达有明显影响，提示这两种生长因子可能以自分泌／旁分泌机制，参与Ｅ２对垂体前叶细胞功能的部分作用     

表达反义细胞周期蛋白cyclinD1载体对胰腺癌细胞生长和凋亡的影响

目的观察反义细胞周期蛋白ｃｙｃｌｉｎＤ１对胰腺癌细胞生长的影响。方法采用Ｓｏｕｔｈｅｒｎｂｌｏｔ和Ｎｏｒｔｈｅｒｎｂｌｏｔ方法检测了５株人胰腺癌细胞系中ｃｙｃｌｉｎＤ１的扩增及表达情况，发现ＰＣ７细胞中基因有扩增及过度表达。我们构建了反义（ＡＳ）ｃｙｃｌｉｎＤ１的表达载体，采用ｌｉｐｏｆｅｃｔｉｎ转染方法转染ＰＣ７细胞，获得转化细胞系ＰＣ７／ＡＳｃｙｃｌｉｎＤ１。经Ｎｏｒｔｈｅｒｎｂｌｏｔ、Ｗｅｓｔｅｒｎｂｌｏｔ检测转化细胞系，表明有外源反义ｃｙｃｌｉｎＤ１的表达，而内源性ｃｙｃｌｉｎＤ１ｍＲＮＡ表达及蛋白合成下调，并进行了细胞凋亡分析。结果转化细胞系细胞恶性生物学行为及表型部分逆转，细胞生长速度、ＤＮＡ合成及细胞增殖和代谢能力均明显下降，软琼脂集落形成能力减低。流式细胞术分析发现细胞被阻滞于Ｇ１期，ＤＮＡ凝胶电泳、原位凋亡检测发现凋亡细胞增多。结论通过反义ＲＮＡ技术，下调在细胞周期调控中起重要作用的ｃｙｃｌｉｎＤ１的表达，可使胰腺癌细胞恶性表型部分逆转。     

人脑胶质母细胞瘤细胞质SHG—44中血管生成因子和细胞周期调 …

OBJECTIVE: To investigate the biological features and immunophenotypes of human glioblastoma cell line SHG-44 after long term passage. METHODS: Immunohistochemistry and in situ hybridization were used to study the proliferative activity, intermediate filament protein coexistence, expressions of oncoprotein, angiogenic factors and cell cycle regulation factors. RESULTS: After 130 to 150 passages, SHG-44 cells were weakly positive for glial fibrillary acidic protein (GFAP), but strongly positive for vimentin. The labeling index of Ki-67 and PCNA were 83.5% +/- 10.2% and 70.0% +/- 18.7% respectively. Overexpression of p21 ras, c-erbB-2, epidermal growth factor (EGF) and EGF receptor were obtained. Basic fibroblast growth factor (bFGF), FGF receptor, vascular endothelial growth factor (VEGF) and inducible nitric oxide synthase (iNOS) were also up-regulated in this cell line. p16, p53, cdk4 and cyclin D1 could be detected in the cells and their indices were 43.1% +/- 11.2%, 20.7% +/- 6.6%, 33.1% +/- 11.4% and 29.2% +/- 4.7% respectively. CONCLUSION: Expressive abnormalities of these growth factors, their receptors and the above oncoproteins as well as disorders of cell cycle regulation contribute to the rapid growth and high degree of malignancy of this cell line.     

Reversal of inhibition of rat glomerular epithelial cell growth by growth factors.

The ability of several growth factors to reverse heparin-induced inhibition of rat glomerular epithelial cell (GEC) growth and the mechanism of growth inhibition were explored in vitro. Insulin-like growth factor-1, rat multiplication-stimulating activity, and platelet-derived growth factor had no effect on proliferation of cultured GEC exposed to heparin (100 micrograms/ml). Epidermal growth factor (EGF) partially reversed heparin-induced growth inhibition in a dose-dependent fashion with a maximum effect seen at 1 ng/ml. No additive effect was seen with combinations of EGF and the other growth factors assayed. A decrease in EGF-stimulated incorporation of 3H-thymidine by GEC was seen with as little as 2 hours of heparin exposure and persisted for up to 48 hours. Heparin consistently increased binding of 125I-EGF to GEC with a significant increase apparent after 2 hours of exposure and a further increase with a 24-hour exposure. Increased EGF binding to heparin-treated cells was due to a significant increase in the association constant of EGF and its receptor with no effect on receptor number. Interactions between GEC and heparinlike glycosaminoglycans in the glomerular basement membrane may play a role in the regulation of GEC proliferation in normal and diseased states.     

旁路信号通路激活介导的EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib继发耐药的研究

目的:本研究旨在探讨肝细胞生长因子(HGF)、表皮生长因子(EGF)及转化生长因子α(TGF-α)是否以旁路激活的方式诱导EML4-ALK融合基因阳性肺癌细胞株H3122对alectinib的耐药,并进一步探讨旁路信号激活在alectinib耐药中的作用。方法:用不同浓度的alectinib、克唑替尼(crizotinib)、17-DMAG或(和)HGF(50μg/L)、EGF(100μg/L)、TGF-α(100μg/L)处理EML4-ALK阳性肺癌细胞株H3122,采用CCK-8法检测细胞活力,流式细胞术检测细胞凋亡,应用Western blot技术检测细胞中ALK、c-Met、EGFR及相应磷酸化蛋白的表达,观察其下游通路关键蛋白AKT、ERK、p-AKT和p-ERK水平。结果:Alectinib作用72 h后,H3122细胞株的活力随着alectinib药物浓度的增加而逐渐下降,呈剂量依赖性。HGF、EGF和TGF-α诱导后,alectinib抑制H3122细胞的生长曲线往右移,HGF、EGF和TGF-α处理能够降低alectinib对肺癌细胞活力的抑制作用。0.05μmol/L alectinib作用H3122细胞株48 h后的凋亡率为(20.12±1.36)%,而alectinib联合HGF、EGF和TGF-α后的凋亡率分别为(7.85±1.03)%、(5.60±0.79)%和(4.58±1.00)%,显著低于alectinib单药处理(P0.05)。Alectinib单药成功抑制p-ALK及其下游信号通路,HGF明显增加细胞中p-Met及其下游p-AKT、p-ERK的蛋白水平,EGF和TGF-α明显增加细胞中p-EGFR及其下游p-AKT、p-ERK的表达,alectinib抑制p-ALK,但不能抑制HGF、EGF和TGF-α诱导的pAKT和p-ERK的蛋白表达。此外,联合应用crizotinib和17-DMAG可以抑制因HGF和EGFR配体而导致的H3122耐药细胞的活力。结论:HGF、EGF和TGF-α可通过旁路激活的方式诱导EML4-ALK阳性肺癌细胞H3122对alectinib耐药,其机制可能与HGF激活c-Met磷酸化、EGF和TGF-α激活EGFR磷酸化有关。     

The effects of growth factors on human normal placental cytotrophoblast cell proliferation

The effects of growth factors were investigated on the proliferation of a normal placental cytotrophoblast cell line (NPC). Epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) and insulin- like growth factor-I (IGF-I) stimulated NPC cell proliferation. In contrast, TGFbeta1 was found to be a negative regulator, inhibiting EGF- induced cell proliferation. When EGF/TGF alpha receptor was analysed by radio-ligand binding, two binding sites of different affinities were revealed in the proliferating NPC cells but only the low affinity binding site was detected in the non-proliferating cytotrophoblast cells in primary cultures. The results suggest that EGF stimulates cytotrophoblast proliferation through high affinity binding sites.      

Characterization of a newly established human acinic cell adenocarcinoma cell line (HACC) originating from the salivary gland: Morphological features and role of various growth factors on the growth of the HACC cell line

Human acinic cell adenocarcinoma cell (HACC) line was established from the pleural effusion that contains meta-static tumor cells of acinic cell adenocarcinoma of papillary and microcystic type originating from the parotid gland. The HACC cells grew in an adherent monolayer with a doubling time of 66 h. Implanted tumor of SCID mice revealed similar histologlcal findings to that of the primary tumor. The HACC cells produced mucin and expressed epithelial markers as well as α₁-antitrypsin and lysozyme, whereas salivary peptide P-C was expressed in cultured HACC cells but not In the primary and Implanted HACC cell tumors. S-100 protein was also expressed in both the primary tumor and HACC cell line. Neither amplification of common oncogenes nor expression of p53 was observed. The receptor for epidermal growth factor (EGF) was expressed, indicating EGF and transforming growth factor-α (TGF-α) enhanced the growth of the HACC line. Unexpectedly, tumor necrosis factor-α (TNF-α) also enhanced the growth of the HACC line significantly. However, there was no evidence of autocrine growth using these growth factors. In contrast, TGF-β1 inhibited the growth of the HACC cell line through apoptosis. The HACC cell line has features similar to both acinar and intercalated ductal cells of the salivary gland. Epidermal growth factor, TGF-α and TNF-α are potential growth factors for the HACC cell line. The HACC cell line may be a good model for studying the biological behavior of salivary gland neoplasms.     

Replicative potential and the duration of the cell cycle in human fibroblasts: coordinate stimulation by epidermal growth factor.

The in vitro replicative potential of human diploid fibroblasts can be increased by polypeptide growth factors such as epidermal growth factor (EGF). Also, the cycle time of EGF-stimulated cells is, on average, decreased and their mitotic cell volume is reduced. Therefore, the regulation of cell size by the duration of the cell cycle may be one process which determines replicative potential. The growth response to a continuous presence of EGF, however, appears to be limited by eventual desensitization to the growth factor.     

Mucin biosynthesis: epidermal growth factor downregulates core 2 enzymes in a human airway adenocarcinoma cell line

Enzymes which exhibit core 2 beta1,6 N-acetylglucosaminyltransferase (C2GnT) activity play important roles in physiologic processes including the inflammatory response and immune system function, and C2GnT activity is regulated during processes, such as T cell activation and cellular differentiation. In this study, we have examined the regulation of C2GnT activity in the H292 airway epithelial cell line by epidermal growth factor (EGF), which has been previously shown to upregulate expression of the airway mucin MUC5AC in this cell line. We found that EGF suppressed C2GnT activity in a time- and dose-dependent fashion, and also suppressed core 4 beta1,6 N-acetylglucosaminyltransferase (C4GnT) activity. Consistent with the suppression of C4GnT activity, Northern blotting results showed that EGF preferentially inhibited the M isoform of C2GnT, which forms core 2, core 4, and blood group I beta1,6 branched carbohydrate structures, while the L isoform, which forms only the core 2 structure, was only modestly affected. Furthermore, EGF treatment resulted in a shift in the carbohydrate structure of FLAG-tagged MUC1 expressed in the cells from core 2-based toward core 1-based structures, consistent with the inhibitory effects of EGF on C2GnT. Transforming growth factor alpha mimicked the effect of EGF on C2GnT, implicating the EGF receptor (EGF-R) in C2GnT suppression, and the EGF-R tyrosine kinase inhibitor AG1478 blocked C2GnT suppression, confirming the role of EGF-R in the inhibition of C2GnT expression. Also, PD98059, a specific inhibitor of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)1/2 in the Ras-mitogen-activated protein kinase pathway, completely blocked the EGF suppressive effect, suggesting possible involvement of the Ras-mitogen-activated protein kinase pathway in EGF-mediated downregulation of C2GnT. The results of this study suggest that exposure of airway cells to EGF may result in remodeling of mucin carbohydrate structure, potentially altering the biological properties of the cells.     

Transcriptional profiling identifies genes induced by hepatocyte-derived extracellular matrix in metastatic human colorectal cancer cell lines

The milieu of the liver, and in particular hepatocyte-derived extracellular matrix (hECM), is a critical factor regulating development of liver metastases of colorectal cancer (CRC) cells. The present study has investigated genes altered by hECM in CRC cells and particularly by heparan sulfate chains of hepatocyte proteoglycans. Gene profiling analysis shows that after 2 days on hECM, 226 genes are up-regulated more than 2-fold in strongly metastatic SM cells, including genes involved in growth arrest and apoptosis, signal transduction, cell migration, proliferation, communication and angiogenesis, with activation of the erbB signaling network and p53 effectors. Genes down-regulated by hECM include genes involved in lipogenesis and the S phase of the cell cycle. Further studies exploring the kinetics of gene expression after 4 and 7 days culture on hECM show induction of EGF family members and of stem cell markers. In particular, hECM, but not collagen, increases mRNA expression of HB-EGF and colon stem cell marker leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5). Expression of these genes is not induced by hECM depleted of the heparan sulfate chains of proteoglycans. Lastly, a specific cell population positive for cancer stem cell (CSC) markers LGR5, epCAM and CD133, but negative for CD44, appears after 7 days culture on hECM, a population which is reduced by 50 % in cells grown on heparan sulfated-depleted hECM. Collectively, the data suggest that hECM induces growth factors and receptors regulating proliferation of metastatic CRC in the liver and offers a growth advantage for specific populations expressing CSC markers.     

Mechanisms of muscle stem cell expansion with cytokines.

Stem cell expansion and proliferation are important for cell transplantation and stem cell-mediated applications. While we have demonstrated that muscle stem cells can be obtained from adult skeletal muscle tissue, these cells represent only a small percentage of the muscle-derived cells and require in vitro expansion for successful stem cell-mediated therapies. In this study, we have examined the potential of several cytokines to stimulate stem cell growth by combining a non-exponential mathematical model with a unique cell culture system. The growth kinetics of two populations of muscle stem cells were characterized in culture medium supplemented with epidermal growth factor (EGF), fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), FLT-3 ligand, hepatocyte growth factor, or stem cell factor (SCF). The division time (DT) and fraction of mitotically active cells were investigated as key parameters to further understand the mechanism of the expansion of the stem cell populations. Our results show that expansion of the freshly isolated, muscle-derived stem cells (MDSC) occurred by recruiting cells into the cell cycle in the presence of EGF, IGF-1, and SCF. However, expansion of the cultured stem cell clone, MC13, is attributed to a reduction of the length of the cell cycle in the presence of FGF-2, EGF, IGF-1, and SCF. Both MDSC and MC13 growth were inhibited in the presence of FLT-3 ligand by increasing the length of the cell cycle. Our results suggest that EGF, IGF-1, FGF-2, and SCF are important cytokines for stimulating the proliferation of MDSC. In addition, this study illustrates that expansion of stem cells occurs through different mechanisms, which consequently demonstrates the importance of monitoring several parameters of cell growth, such as DT and dividing fraction, following stimulation with growth factors.     

Growth factors and cytokines in paragangliomas and pheochromocytomas,with special reference to sustentacular cells

The aim of this study was to localize various growth factors and cytokines in paragangliomas and pheochromocytomas in order to understand their possible autocrine or paracrine functions, and to compare sustentacular cells of the adrenal medulla with pituitary stellate cells. Thirteen resected tumors, 11 paragangliomas and 2 pheochromocytomas of the adrenal medulla, were studied. In addition, five surgically removed nontumorous adrenals and five nontumorous pituitaries were studied. Varying numbers of sustentacular cells were immunopositive for S-100 protein and in most instances for glial fibrillary acidic protein. Insulin-like growth factor-1 (IGF-1), tumor necrosis factor-α (TNF-α), and interleukin-6 were localized to both cell types in all cases, whereas epidermal growth factor (EGF) immunopositivity was noted in only three. In all tumors, leukemia inhibitory factor (LIF) was restricted to chief cells and EGF receptor to sustentacular cells. Nontumorous chief cells and sustentacular cells of adrenal medulla exhibited immunoreactivities similar to those of paragangliomas and pheochromocytomas. Secretory adenohypophysial cells displayed various immunoreactivities for all growth factors, receptors, and cytokines studied. Pituitary stellate cells were immunopositive for EGF, EGF receptor, IGF-1, LIF, and TNF-α. In conclusion, paragangliomas and pheochromocytomas are immunoreactive for a wide spectrum of growth factors and cytokines. Immunocytochemistry demonstrated similarities between sustentacular cells and stellate cells of the pituitary in addition to their similar morphology. The significance of these observations regarding paracrine activities of chief and sustentacular cells remains to be determined.