Clinical significance of upregulated Rho GTPase activating protein 12 causing resistance to tyrosine kinase inhibitors in hepatocellular carcinoma

BACKGROUND Hepatocellular carcinoma(HCC)is a major health challenge with high incidence and poor survival rates in China.Systemic therapies,particularly tyrosine kinase inhibitors(TKIs),are the first-line treatment for advanced HCC,but resistance is common.The Rho GTPase family member Rho GTPase activating protein 12(ARHGAP12),which regulates cell adhesion and invasion,is a potential therapeutic target for overcoming TKI resistance in HCC.However,no studies on the expression of ARHGAP12 in HCC and its role in resistance to TKIs have been reported.AIM To unveil the expression of ARHGAP12 in HCC,its role in TKI resistance and its potential associated pathways.METHODS This study used single-cell RNA sequencing(scRNA-seq)to evaluate ARHGAP12 mRNA levels and explored its mechanisms through enrichment analysis.CellChat was used to investigate focal adhesion(FA)pathway regulation.We integrated bulk RNA data(RNA-seq and microarray),immunohistochemistry and proteomics to analyze ARHGAP12 mRNA and protein levels,correlating with clinical outcomes.We assessed ARHGAP12 expression in TKI-resistant HCC,integrated conventional HCC to explore its mechanism,identified intersecting FA pathway genes with scRNA-seq data and evaluated its response to TKI and immunotherapy.RESULTS ARHGAP12 mRNA was found to be highly expressed in malignant hepatocytes and to regulate FA.In malignant hepatocytes in high-score FA groups,MDK-[integrin alpha 6(ITGA6)+integrinβ-1(ITGB1)]showed specificity in ligand-receptor interactions.ARHGAP12 mRNA and protein were upregulated in bulk RNA,immunohistochemistry and proteomics,and higher expression was associated with a worse prognosis.ARHGAP12 was also found to be a TKI resistance gene that regulated the FA pathway.ITGB1 was identified as a crossover gene in the FA pathway in both scRNA-seq and bulk RNA.High expression of ARHGAP12 was associated with adverse reactions to sorafenib,cabozantinib and regorafenib,but not to immunotherapy.CONCLUSION ARHGAP12 expression is elevated in HCC and TKI-resistant HCC,and its regulatory role in FA may underlie the TKI-resistant phenotype.

Modulatory Effect of Rg_1 on the Activity of ProteinTyrosine Kinase of Lymphocytes in the Elderly

The effect of Rg1,a saponin extracted froin Panax ginseng, on the phenotype,receptor and the activity of protein tyrosine kinase (PTK) of lymphocytes isolated from 7 healthy oldpersons were studied. The CD25, CD45RA and CD45RO phenotypes of lymphocytes were 4eter-mined by indirect immunofluorescence technique. The percentage of CD25, CD45RA and CD45ROpositive lymphocytes was 38.3%±17.3%, 46.0% 15.1%, and 52.6%±14.1% respectively after incu-bation with PHA (5 μ±/ml) for 72 hours. However, there were 58.0%±12.5%, CD25, 64.1% ± 12.4%,CD45RA, and 74.0%±8.0%, CD45RO positive cells in the presence of Rg, ( 1μg/ml) along with PHA(5 μg/ml) over the sanie period of incubation. A significant increase was induced by Rgi (P<0.05).The activities of PTK in the cytoplasm and membrane of lymphocytes were measured by ELISAmcthod after incubation with PHA or PHA+Rg1. The absorbance value of PTK activity in cytoplasmafter 72 hr incubation was 0. 120±0.020 in PHA group, but 0. 1 38±0.015 in PHA+Rg1 group. In thelymphocyte membrane, it was 0.374± 0.060 in PHA group and 0.403 ± 0.008 in PHA+Rg1 group(P<0.001). These results showed that Rgi significantly arid simultaneously increased both the PT Kactivity and the expression of phenotype of lymphocytes.

EFFECT OF ACTIVE COMPOUNDS ISOLATED FROM PTERIS SEMIPINNATA L ON DNA TOPOISOMERASES AND TYROSINE PROTEIN KINASE AND EXPRESSION OF C-MYC IN LUNG ADENOCARCINOMA CELLS

Objective: To study the effect of active compound 6F and A from Pteris semipinnata L.(PsL) on the activities of DNA topoisomerase (TOPO) I and II, activities of cytosolic and membrane TPK, and expression of oncogene c-myc in lung adenocarcinoma cells. Methods: The effect of compound 6F and A on activities of cytosolic and membrane TPK was measured by scintillation counting; the effect of compound A on expression of oncogene c-myc was determined by flow cytometry indirect fluorimetry. Results: compound 6F and A could inhibit the activities of TOPO I, and they strongly inhibited the TOPO II in 0.01 mg/L and 10.0 mg/L respectively. Compound A slightly inhibited the activities of membrane TPK, but not the cytosolic one. Compound A could inhibit the expression of oncogene c-myc. Conclusion: Topoisomerases are target of compound 6F and A. Compound A could slightly inhibit the activities of TPK, and showed an inhibitory effect on the expression of oncogene c-myc.

MicroRNA-298 determines the radio-resistance of colorectal cancer cells by directly targeting human dual-specificity tyrosine(Y)-regulated kinase 1A

BACKGROUND Radiotherapy stands as a promising therapeutic modality for colorectal cancer(CRC);yet,the formidable challenge posed by radio-resistance significantly undermines its efficacy in achieving CRC remission.AIM To elucidate the role played by microRNA-298(miR-298)in CRC radio-resistance.METHODS To establish a radio-resistant CRC cell line,HT-29 cells underwent exposure to 5 gray ionizing radiation that was followed by a 7-d recovery period.The quantification of miR-298 levels within CRC cells was conducted through quantitative RT-PCR,and protein expression determination was realized through Western blotting.Cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and proliferation by clonogenic assay.Radio-induced apoptosis was discerned through flow cytometry analysis.RESULTS We observed a marked upregulation of miR-298 in radio-resistant CRC cells.MiR-298 emerged as a key determinant of cell survival following radiation exposure,as its overexpression led to a notable reduction in radiation-induced apoptosis.Intriguingly,miR-298 expression exhibited a strong correlation with CRC cell viability.Further investigation unveiled human dual-specificity tyrosine(Y)-regulated kinase 1A(DYRK1A)as miR-298’s direct target.CONCLUSION Taken together,our findings underline the role played by miR-298 in bolstering radio-resistance in CRC cells by means of DYRK1A downregulation,thereby positioning miR-298 as a promising candidate for mitigating radioresistance in CRC.

ABNORMAL PROTEIN TYROSINE KINASES ASSOCIATED WITH HUMAN HAEMATOLOGICAL MALIGNANCIES

Objective: To survey the role of protein tyrosine kinases (PTKs) in the pathogenesis of several hematopoietic malignancies. Methods: By reviewing the published laboratory and clinical studies on PTK-related oncoproteins and their causative role in some leukemias and lymphomas. Results: Protein tyrosine kinases are key participants in signal transduction pathways that regulate cellular growth, activation and differentiations. Aberrant PTK activity resulting from gene mutation (often accompanying chromosome translocation) plays an etiologic role in several clonal hematopoietic malignancies. For example, the PTK product of the BCR-ABL fusion gene resulting from the t (9; 22) translocation exhibits several fold higher tyrosine kinase activity than the product of the ABL gene. Evidence suggests that the BCR-ABL oncoprotein alone is sufficient to case chronic myelogenous leukemia (CML) and other Ph positive acute leukemia. PTK over-activity resulting from chromosomal translocations creating TEL-ABL, TEL-JAK2 and TEL-PDGFRβ fusion proteins plays an important role in the pathogenesis of other types of leukemia. Another example occurs in anaplastic large cell lymphoma (ALCL). Experimental and clinical evidences indicate that translocations involving ALK gene on chromosome 2p23, most commonly resulting in an NPM-ALK fusion oncogene, result in constitutive activation of ALK and cause ALCL. This group of lymphomas is now named ALK positive lymphoma or ALKoma. Conclusion: Genetic lesions creating aberrant fusion proteins that result in excessive PTK activity are increasingly being recognized as central to the pathogenesis of hemotopoietic malignancies. These chimeric PTK molecules represent attractive disease-specific targets against which new classes therapeutic agents are being developed.

Effects of insulin receptor tyrosine protein kinase on insulin resistance after scalding in rats

After the rats were inflicted with 30% TBSA full thickness scalding on their back, the changes of insulin receptor tyrosine protein kinase (IRTPK) were studied on the 4th day postscalding with the phosphorylation of exogenous substrates stimulated with hepatic and muscular plasma membrane in order to explore the possible molecular mechanism of insulin resistance after thermal injury. It was found that insulin resistance appeared in the early stage postscalding while the basic value of phosphorylation of exogenous substrates stimulated with hepatic and muscular plasma membrane was increased and the activity of IPTPK to insulin stimulation was greatly suppressed as compared with the control. These facts suggest that the impaired responses of IRTPK to insulin stimulation may affect the down-stream signal transmission of the insulin receptors and then the signal coupling pathwayfor glucose transmembranous transportation and glycogen synthesis mediated by IRTPK is obstructed and insulinresistance develops.

Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

AIM:To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration. METHODS:Gastric lesions were induced in rats using restraint cold stress.To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair,total activity of protein tyrosine kinase(PTK),protein tyrosine phosphatase (PTP),antioxidant enzymes,nitric oxide synthase (NOS), 2',5'-oligoadenylate synthetase,hydroxyl radical and zinc levels were assayed in parallel. RESULTS:Ulcer provocation induced an immediate decrease in tyrosine kinase(40% in plasma membranes and 56% in cytosol,(P<0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol),followed by 2.3-2.4-fold decrease (P<0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity,30% increase (P<0.05) in catalase activity,2.3-fold inhibition (P<0.05) of glutathione peroxidase,3.3-fold increase (P<0.05) in hydroxyl radical content,and 2.3-fold decrease (P<0.05) in zinc level in gastric mucosa.NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration,PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase,P<0.05),but remained inhibited(1.6-3-fold decrease on days 3,4 and 5,P<0.05) in the cytosol.Tyrosine phosphatases remained inhibited both in membranes and cytosol(1.5-2.4-fold,P< 0.05).NOS activity remained increased on days 1,2 and 3(3.8-,2.6-,2.2-fold,respectively,P<0.05).Activity of SOD increased 1.6 times(P<0.05)days 4 and 5 after stress.Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3-and 2-fold,respectively,P<0.05)on the last day. Activity of 2',5'-oligoadenylate synthethase increased 2.8-fold (P<0.05) at the beginning,and 1.6-2.3-fold (P<0.05) during ulcer recuperation,and normalized on day 5,consistent with slowing of inflammation processes. CONCLUSION:These studies show diverse changes in total tyrosine kinase activity in gastric mucosa during the recovery process.Oxidative and nitrosative stress during lesion formation might lead to the observed reduction in tyrosine phosphorylation during ulceration.

Protein tyrosine phosphatase 1B regulates migration of ARPE-19 cells through EGFR/ERK signaling pathway

AIMTo evaluate whether protein tyrosine phosphatase 1B (PTP1B) contributed to initiate human retinal pigment epithelium cells (A)-19 migration and investigate the signaling pathways involved in this process.METHODSARPE-19 cells were cultured and treated with the siRNA-PTP1B. Expression of PTP1B was confirmed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). AG1478 [a selective inhibitor of epidermal growth factor receptor (EGFR)] and PD98059 (a specific inhibitor of the activation of mitogen-activated protein kinase) were used to help to determine the PTP1B signaling mechanism. Western blot analysis verified expression of EGFR and extracellular signal-regulated kinase (ERK) in ARPE-19 cells. The effect of siRNA-PTP1B on cell differentiation was confirmed by immunostaining for &#x003b1;-smooth muscle actin (&#x003b1;-SMA) and qRT-PCR. Cell migration ability was analyzed by transwell chamber assay.RESULTSThe mRNA levels of PTP1B were reduced by siRNA-PTP1B as determined by qRT-PCR assay. SiRNA-PTP1B activated EGFR and ERK phosphorylation. &#x003b1;-SMA staining and qRT-PCR assay demonstrated that siRNA-PTP1B induced retinal pigment epithelium (RPE) cells to differentiate toward better contractility and motility. Transwell chamber assay proved that PTP1B inhibition improved migration activity of RPE cells. Treatment with AG1478 and PD98059 abolished siRNA-PTP1B-induced activation of EGFR and ERK, &#x003b1;-SMA expression and cell migration.CONCLUSIONPTP1B inhibition promoted myofibroblast differentiation and migration of ARPE-19 cells, and EGFR/ERK signaling pathway played important role in migration process.

机械应力调控血管平滑肌细胞的凋亡

背景:随着生物力学的发展,其在心血管疾病中的研究也日益广泛,通过研究血管的力学特性可以有效地揭示动脉粥样硬化、再狭窄等心血管疾病的发病机制,开发心血管疾病治疗的新思路和新方法。目的:综述机械应力刺激对血管平滑肌细胞凋亡的研究现状,寻找临床治疗潜在靶分子和信号通路,以期改善临床动脉粥样硬化及再狭窄等心血管疾病的临床治疗效果。方法:检索1992年1月至2023年5月在中国知网、PubMed及ScienceDirect数据库收录的文献。中文检索词为“血管平滑肌细胞,机械应力,剪切力,牵张力,凋亡”;英文检索词为“vascular smooth muscle cell,mechanical stress,shear stress,stretch stress,apoptosis”,最终纳入63篇文献进行综述分析。结果与结论:①血管平滑肌细胞的生理性和病理性凋亡都是为了适应血管力学变化而发生的适应性重构。不同部位的平滑肌细胞承受不同的力学刺激,其发病机制也不同。②低剪切力、生理剪切力和高剪切力可通过直接与平滑肌细胞表面分子、受体及蛋白等作用调控凋亡信号分子和抑制增殖实现对血管平滑肌细胞凋亡的调控,该部分未对促进增殖的研究进行综述。③低牵张力、生理牵张力和高牵张力可导致平滑肌细胞凋亡,但仍存在争议。平滑肌表面存在多种力学感受分子(如整合素及受体络氨酸激酶等)可以将力学信号转导为细胞内的化学信号(如Hippo通路),启动平滑肌细胞的凋亡信号,调控平滑肌细胞的凋亡。④总之,不同力学刺激通过多种信号分子,启动多个信号通路共同作用,调控平滑肌细胞的凋亡,例如剪切力主要通过刺激分泌前列腺素、转化生长因子等影响Fas/FasL、Akt通路;而牵张力主要通过Yes相关蛋白等调控YAP通路和Notch通路等。这些分子在不同的作用时间,或不同作用强度下可能发挥相反的双向作用,比如,小鼠血管平滑肌细胞受到10%生理牵张1 h时,其增殖增加;然而,人的血管平滑肌细胞牵张12 h后可以减少其增殖。临床可以通过寻找其关键作用时间点干扰力学转导关键分子,达到预防和治疗临床心血管疾病的目的。

半边旗有效化合物对肺癌细胞DNA拓扑异构酶、TPK及c-myc基因的影响

研究半边旗有效化合物6F和A对肺癌细胞DNA拓扑异构酶(TOPO)Ⅰ和Ⅱ活性的影响;化合物A对细胞胞浆和胞膜酪氨酸蛋白激酶(TPK)活性及癌基因c -myc蛋白表达的影响。方法 :通过电泳法测定化合物6F和A对肺癌细胞DNA拓扑异构酶(TOPO)Ⅰ、Ⅱ活性的影响;用液体闪烁计数法测定化合物A对酪氨酸蛋白激酶(TPK)活性的影响;流式细胞仪间接荧光检测法测定化合物A对c -myc基因蛋白表达的影响。结果 :化合物6F和A可抑制细胞DNA拓扑异构酶Ⅰ、Ⅱ的活性,1 0mg/L的6F和A即开始抑制TOPOⅠ的活性,随浓度升高抑制作用增强 ;0 01mg/L的6F和10 0mg/L的A对TOPOⅡ的活性有强烈的抑制作用;化合物A对细胞中胞膜TPK活性有一定的抑制作用,但对胞浆TPK的活性几乎没有影响;化合物A对c -myc基因的蛋白表达有抑制作用。结论 :DNA拓扑异构酶是化合物6F和A抑制细胞生长的靶点之一,化合物A对TPK活性有一定的抑制作用,对c

PKC、PKA和TPK在血小板激活中的作用

利用^（３２）Ｐ－ＮａＨ_２ＰＯ_４标记猪血小板，然后以ＰＭＡ、凝血酶、ＰＧＥ_１、腺苷等处理，结果表明，随着ＰＭＡ激活ＰＫＣ，血小板发生聚集。３５μｍｏｌ／ＬＰＧＥ_１或１ｍｍｏｌ／ＬｄｂｃＡＭＰ不能抑制５０ｎｍｏｌ／ＬＰＭＡ诱导的血小板聚集，腺苷却能抑制ＰＭＡ诱导的血小板聚集（ＥＣ_（５０）＝０．１ｍｍｏｌ／Ｌ），ｄｂ－ｃＡＭＰ、腺苷都不能抑制１００ｎｍｏｌ／ＬＰＭＡ诱导的４０ｋＤ蛋白磷酸化。ＰＫＡ激活不能抑制ＰＭＡ激活的ＰＫＣ。在ＰＭＡ、凝血酶激活的血小板中，ＰＫＣ、ＴＰＫ都发生激活，４０ｋＤ底物既是ＰＫＣ的底物又是ＴＰＫ的底物，ＰＫＣ和ＴＰＫ在血小板聚集中起着重要的调节作用。

钒酸钠改善严重烫伤大鼠肌细胞胰岛素受体TPK活性的研究

目的：探讨钒酸钠改善创伤后胰岛素抵抗和糖代谢紊乱的机制。方法：用钒酸钠胃饲３０％ＴＢ－ＳＡⅢ度烫伤大鼠，观察血糖、胰岛素、肌细胞膜胰岛素受体结合特性和胰岛素受体酪氨酸蛋白激酶（ＩＲ－ＴＰＫ）活性的改变。结果：钒酸钠降低严重烫伤大鼠空腹血糖水平，不改变胰岛素水平，也不改变肌细胞膜胰岛素受体最大结合率和结合容量，但可显著增加胰岛素刺激的ＩＲ－ＴＰＫ活性。结论：钒酸钠可不通过胰岛素受体介导而直接刺激ＩＲ－ＴＰＫ活性，改善靶组织对胰岛素的敏感性。

体外循环和非体外循环病人胰岛素受体TPK活性及血浆TNF-α变化及意义

目的 观察体外循环和非体外循环手术中骨骼肌胰岛素受体TPK活性及血浆TNF α改变 ,探讨胰岛素抵抗产生的机制。方法 随机选择 8例动脉导管患者及 1 5例房缺或室缺患者 ,于麻醉前、麻醉后、开胸后 2 0min、术毕、术后 6h采取血样 ,测定血糖、胰岛素、TNF α的浓度 ,并于手术开始即刻、术毕关胸即刻取骨骼肌标本 ,测定胰岛素受体酪氨酸蛋白激酶 (TPK)活性的变化。结果 体外循环组血糖、胰岛素、TNF α均明显升高 ,手术结束时骨骼肌胰岛素受体TPK活性明显低于手术开始时。动脉导管组血糖明显升高 ,胰岛素及TNF α有所升高 ,但不显著 ,手术前后骨骼肌胰岛素受体TPK活性无明显改变。结论 体外循环手术机体胰岛素抵抗程度明显强于非体外循环手术 ,体外循环可作为一个独立损伤因素影响胰岛素受体TPK活性从而干扰了胰岛素的正常信号传导 ,产生明显的胰岛素抵抗 ,TNF

aFGF对肺腺癌AGZY-83A细胞株TPK、PKC活性及Ca^(2+)浓度的影响

目的：观察ａＦＧＦ与细胞膜上特异受体结合后引起的细胞内信号转导途径，探讨ａＦＧＦ导致细胞增殖的机理。方法：以不同浓度的ａＦＧＦ处理ＡＧＺＹ－８３Ａ细胞，利用［γ－３２Ｐ］ＡＴＰ掺入外源性底物的方法测定受体的酪氨酸蛋白激酶活性（ＴＰＫ）及蛋白激酶Ｃ（ＰＫＣ）活性；用Ｆｕｒａ－２／ＡＭ为荧光指示剂测定［Ｃａ２＋］ｉ。结果：随着ａＦＧＦ浓度增加，ＴＰＫ及ＰＫＣ活性随之升高。当ａＦＧＦ浓度为１．１２μｇ／ｍｌ时ａＦＧＦ处理组的ＴＰＫ是对照组的４倍；膜ＰＫＣ活性也是对照组的４倍，胞浆ＰＫＣ活性是对照组的１．７５倍。［Ｃａ２＋］ｉ是对照组的３倍。结论：该细胞株中ａＦＧＦ受体具有ＴＰＫ活性。ＴＰＫ激活后进一步促进蛋白质和酶磷酸化，而使ＰＫＣ活性及［Ｃａ２＋］ｉ升高，即ＰＫＣ和Ｃａ２＋是ＴＰＫ的下游信号分子，进一步促进ｃ－ｆｏｓ、ｊｕｎ基因表达增加。

两株鼻咽癌细胞中TPK、PKC的活性研究

目的：研究ＣＮＥ－１和ＣＮＥ－２Ｚ细胞浆中ＰＫＣ、ＴＰＫ及胞膜ＴＰＫ活性，及槲皮素对它们的影响；方法：常规培养ＣＮＥ－１、ＣＮＥ－２Ｚ和淋巴细胞，用特异底物法、特异激活剂法分别测定其ＴＰＫ、ＰＫＣ活性，两两比较用ｔ检验；结果：ＣＮＥ－１细胞胞浆ＰＫＣ、ＴＰＫ均明显高于脐带血淋巴细胞（Ｐ＜０．０１），而胞膜ＴＰＫ无明显变化；ＣＮＥ－２Ｚ细胞胞浆ＰＫＣ却明显低于淋巴细胞（Ｐ＜０．０１），胞浆、胞膜ＴＰＫ活性均明显增高（Ｐ＜０．０１）。槲皮素（１００μｍｏｌ／Ｌ）使ＣＮＥ－２Ｚ细胞中胞膜ＴＰＫ明显的增高（Ｐ＜０．０１），而对其胞浆ＰＫＣ、ＴＰＫ均无明显影响；该浓度下的槲皮素也能使ＣＮＥ－１细胞中胞膜ＴＰＫ活性明显升高（Ｐ＜０．０１），但强烈抑制其胞浆ＴＰＫ活性（Ｐ＜０．０１），而对其胞浆ＰＫＣ也没有明显影响；结论：胞浆。

DHA复合物对小鼠H_(22)肝癌细胞TPK活性影响

目的研究二十二碳六烯酸(DHA)复合物抗癌作用机制。方法以移植H22肝癌细胞的小鼠为模型,利用[γ-32P]三磷酸腺苷(ATP)作为反应启动剂掺入外源性底物的方法测定酪氨酸蛋白激酶(TPK)活性,观察DHA复合物对H22癌细胞内TPK活性的影响。结果DHA复合物的体内抑瘤率可高达70%以上;癌细胞细胞核、细胞质、细胞膜、内质网、线粒体中TPK的活性普遍升高(P<0.01)。给予DHA复合物作用后,癌细胞上述各组分中TPK的活性均显著下降(P<0.01)。结论DHA复合物具有抑制TPK活性,降低细胞内的信号传递,抑制癌细胞增殖的作用。

bFGF及TPK抑制剂对听神经瘤细胞增殖的影响

目的 研究碱性成纤维细胞生长因子(bFGF)和酪氨酸蛋白激酶(TPK)抑制剂对培养的听神经瘤细胞增殖和DNA合成的影响作用,初步探讨其作用机制。方法 选取10例手术切除的新鲜听神经瘤组织若干,常规细胞培养,细胞种植于含10％胎牛血清的培养基中,早期传代细胞用于下一步试验。通过细胞计数和3H-TdR摄取率测定观察bFGF及TPK抑制剂genestein对听神经瘤细胞增殖及DNA合成的影响。结果 在无血清培养条件下,所测10例标本中应用bFGF有9例(9/10)测得的活细胞数和3H-TdR摄取率较对照组显著增加(P<0.05)。genistein在4例标本中对瘤细胞增殖和DNA合成均产生明显抑制效应(P<0.01),亦可阻抑bFGF的促增殖作用(P<0.01)。结论bFGF可能以自或旁分泌形式参与听神经瘤细胞增殖的调节。TPK抑制剂在体外能有效抑制听神经瘤细胞增殖,至少部分是通过抑制bFGF受体功能来实现的。

aFGF对人脐静脉内皮细胞TPK、PKC活性及Ca^(2+)浓度的影响

为了观察酸性成纤维细胞生长因子 ( acidic fibroblast growth factor,a FGF)与人脐静脉内皮细胞 ( human umbilical vein endothelial cell,HUVEC)膜上特异受体结合后引起的细胞内信号转导途径 ,探讨 a FGF导致细胞增殖的机理 ,经 Scatchard曲线分析人脐静脉内皮细胞膜受体性质 .以不同浓度的 a FGF处理人脐静脉内皮细胞 ,利用 [γ- 3 2 P]ATP参入外源性底物的方法测定受体的酪氨酸蛋白激酶 ( tyrosine protein kinase,TPK)及蛋白激酶 C( protein kinase C,PKC)的活性 ;用 Fura-2 /AM为荧光指示剂测定 [Ca2 + ]i.结果显示 :Scatchard曲线证明 a FGF与 HUVEC膜受体特异结合呈一条曲线 ,即受体为一种结合位点 ,Kd 为 3.6× 1 0 -10～ 9.6× 1 0 -10 mol/L,每个细胞受体数为2 70 90 .随着 a FGF浓度增加 ,TPK及 PKC活性随之升高 .当 a FGF浓度为 1 .1 2 mg/L时 ,a FGF处理组的 TPK活性是对照组的 3倍 ;膜 PKC活性是对照组 3.4倍 ,胞浆 PKC活性是对照组的 1 .87倍 .胞浆 [Ca2 + ]是对照组的 3倍 .结果指出 :该细胞中 a FGF受体具有 TPK活性 .TPK激活后进一步促进蛋白质和酶磷酸化级联反应 ,而使 PKC活性及 [Ca2 + ]i 升高 ,即 PKC和 Ca2 + 为 TPK的下游信号分子 ,进一步促进基因表达增加 ,导致细胞增殖 .

HL－60细胞诱导分化过程中TPK和PTPP活力的时空改变

对ＲＡ、ＨＨＴ和ＷＰ_（８５２）诱导ＨＬ－６０细胞分化过程中胞浆和膜溶脱部分中的ＴＰＫ和ＰＴＰＰ活力变化进行了研究．结果表明，在诱导早期，ＴＰＫ的活力就有明显的波动变化；随着诱导时间的延长，胞浆部分ＴＰＫ活力下降，膜溶脱部分的ＴＰＫ活力则升高．诱导后，胞浆部分和膜溶脱部分的ＰＴＰＰ活力均明显升高。与ＴＰＫ活力变化相比较，ＰＴＰＰ变化幅度比ＴＰＫ的大。

bFGF对NIH3T3细胞TPK,PKC,ERK活性的影响

目的 :观察碱性成纤维细胞生长因子 (bFGF)对NIH3T3细胞酪氨酸蛋白激酶 (TPK)、蛋白激酶C(PKC)及胞外调节蛋白激酶 (ERK)活性的影响 ,探讨其信号转导机制。方法 :分别加入bFGF及PKC抑制剂H 7、MEK1抑制剂PD980 5 9孵育细胞 ,利用 [γ 3 2 P]ATP掺入外源性底物的方法测定活性。结果 :bFGF可使细胞膜TPK、细胞质PKC及ERK活性明显升高。H 7+bFGF、PD980 5 9+bFGF组与只加bFGF组比较TPK活性保持不变 ,而PKC及ERK活性均下降。结论 :TPK、PKC、ERK介导bFGF的信号转导。ERK与PKC是TPK受体下游的两个分支 。