苗药九仙罗汉接骨汤含药血清对MC3T3-E1Subclone14细胞增殖、凋亡和Runx2、Osterix、Bax、Bcl-2表达的影响

目的:观察苗药九仙罗汉接骨汤含药血清对MC3T3-E1Subclone14细胞增殖、凋亡及Runx2、Osterix、Bax、Bcl-2 mRNA及蛋白表达的影响,探讨其促进成骨细胞增殖,抑制成骨细胞凋亡的可能机制。方法:将体外培养的MC3T3-E1Subclone14细胞系分为空白对照组、仙灵骨葆胶囊组及九仙罗汉接骨汤含药血清低、中、高浓度组,各组制备含药血清后均用高糖DMEM培养液分别配制成不同浓度进行培养;培养24 h后采用MTT与流式细胞仪检测细胞增殖及凋亡;RT-PCR与Western Blot、免疫组化法测定Runx2、Osterix、Bax、Bcl-2 mRNA及蛋白表达。结果:与空白对照组比较,九仙罗汉接骨汤含药血清中、高浓度组增殖能力显著升高,细胞凋亡率显著降低,Runx2、Osterix、Bcl-2 mRNA及蛋白表达显著升高,Bax mRNA及蛋白表达显著降低(P<0.05或P<0.01)。结论:苗药九仙罗汉接骨汤可促进MC3T3-E1Subclone14细胞增殖,并抑制其凋亡,其作用机制可能与调节Runx2、Osterix、Bax、Bcl-2基因及蛋白表达有关。

EXPRESSING HUMAN MATURED BRAIN-DERIVED NEUROTROPHIC FACTOR GENE IN E.Coli AND DETERMINING ITS BIOACTIVITY

Objective Expressing the human matured brain-derived neurotrophic factor (mBDNF) gene in E. Coli and determining its bioactivity. Methods The resulting gene of mBDNF was subcloned into the EcoRI-BamHI site or the expression vector plasmid pBV220. The ligation products were used to transform the competent E. Coli DH5a. The proteins or mBDNF were experessed by temperature inducing. The expression products were dealed with solubilizing inclusion bodies and refolding protein. It was introduced into the embryonic chicken DRG to test whether the expressed mBDNF is a biologically active protein. Results The recombinant plasmid pBV/mBDNF was success- fully constructed. By temperature inducing, under the control of the bacteriophage λPL promoter, the experessed mBDNF protein was a 14Kd non-fusion protein,which existed in E. Coli as inclusion bodies. The size or expressed mBDNF is identical to the prediction. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of embryonic 8-day-old chicken DRG neurons as compared to control. Conclusion Tke mBDNF gene can be expressed bioactively in E. Coli.

Expression Level of u-PAR in Different Invasive PC-3M Cell Subclones

Objective: To select a target molecule associated with invasive potential in PC-3M cell. Methods: Cell subclones were isolated from PC-3M cell line with the method of limited dilution and their invasive ability characterized by monolayer invasion assay. The expression of u-PAR in the cell subclones at mRNA and protein levels was assayed respectively by non-competitive quantitative RT-PCR and immunohistochemical assay. Results: The expression level of u-PAR in highly invasive cell subclones was higher than that of lower invasive subclones. Conclusion: The higher expression level of u-PAR is associated with the relative strong invasive ability of PC-3M subclones. It is indicated that the u-PAR might be a promising target molecule for inhibiting invasion of highly invasive PC-3M cell subclones.

MC3T3-E1Subclone14体外诱导成骨模型的建立及Ano5基因表达的研究

目的建立MC3T3-E1 Subclone 14体外诱导成骨模型,探讨Ano5基因表达与成骨细胞形成的关系,了解其对成骨分化的影响。方法 MC3T3-E1 Subclone 14细胞贴壁培养,加入条件培养基,分别培养至0d、3d、7d、14d、21d。倒置显微镜下观察细胞形态,通过碱性磷酸酶活力测定和茜素红染色,鉴定成骨细胞。利用REAL TIME-PCR检测Ocn、Colα1、Opg/Rankl、Osterix和Runx2等成骨相关因子的基因表达水平,同时检测Ano5基因在成骨细胞形成过程中的表达趋势。结果体外诱导MC3T3-E1 Subclone 14倒置光学显微镜下观察细胞形态呈梭形。成骨细胞碱性磷酸酶活力逐渐增高。成骨诱导培养至14d和21d,茜素红染色可见细胞表面出现矿化结节。成骨相关因子基因表达均逐渐增高,至21d达到高峰。Ano5基因从诱导分化的第3d开始表达,随后表达量逐渐增高,至14d达到高峰,21d表达量下降。结论在MC3T3-E1 Subclone 14体外诱导分化为成骨细胞过程中,Ano5基因表达量逐渐升高,至14d达到高峰,21d开始表达量具有下降趋势。

MC3T3-E1Subclone14体外诱导成骨细胞模型的建立

目的 建立MC3T3-E1 Subclone 14体外诱导成骨细胞模型,为进一步了解成骨细胞形成机制打下实验基础.方法 MC3T3-E1 Subclone 14细胞培养至贴壁,加入条件培养基：L-抗坏血酸（50μg/mL）、β-甘油磷酸钠（10mmol/mL）、地塞米松（10-8mol/mL）,培养至21d.倒置显微镜观察细胞形态;碱性磷酸酶（ALP）、Von Kossa染色鉴定成骨细胞和骨细胞;RT-PCR、Western-blot检测成骨细胞相关基因骨钙素、骨唾液酸蛋白、骨桥蛋白等相关生物学指标mRNA和蛋白表达水平,以鉴定成骨细胞体外形成.结果 1.体外诱导MC3T3-E1 Subclone 14至7d,倒置显微镜观察细胞成梭形;2.10d ALP染色结果显示：成骨细胞呈红色;3.14d Von Kossa染色结果显示：细胞表面有矿化结节形成;4.相关基因骨钙素,骨桥蛋白,骨唾液酸蛋白的mRNA和蛋白水平在14d均发生变化.结论 成功建立了MC3T3-E1 Subclone 14体外诱导成骨细胞形成模型.

Hunting down the dominating subclone of cancer stem cells as a potential new therapeutic target in multiple myeloma: An artificial intelligence perspective

The development of single-cell subclones,which can rapidly switch from dormant to dominant subclones,occur in the natural pathophysiology of multiple myeloma(MM)but is often"pressed"by the standard treatment of MM.These emerging subclones present a challenge,providing reservoirs for chemoresistant mutations.Technological advancement is required to track MM subclonal changes,as understanding MM's mechanism of evolution at the cellular level can prompt the development of new targeted ways of treating this disease.Current methods to study the evolution of subclones in MM rely on technologies capable of phenotypically and genotypically characterizing plasma cells,which include immunohistochemistry,flow cytometry,or cytogenetics.Still,all of these technologies may be limited by the sensitivity for picking up rare events.In contrast,more incisive methods such as RNA sequencing,comparative genomic hybridization,or whole-genome sequencing are not yet commonly used in clinical practice.Here we introduce the epidemiological diagnosis and prognosis of MM and review current methods for evaluating MM subclone evolution,such as minimal residual disease/multiparametric flow cytometry/next-generation sequencing,and their respective advantages and disadvantages.In addition,we propose our new single-cell method of evaluation to understand MM's mechanism of evolution at the molecular and cellular level and to prompt the development of new targeted ways of treating this disease,which has a broad prospect.

MICROARRAY ANALYSIS OF DIFFERENT GENE EXPRESSION OF HUMAN CERVICAL CANCER SUBCLONE CELL LINES

Objective To examine the differentially expressed invasion-related genes in two anchorage-independent uterine cervical carcinoma cell lines derived from the same patient using a cDNA array. Methods Two human uterine cervical carcinoma subclonal cell lines CS03 and CS07 derived from a single donor line CS1213 were established by limited dilution procedure. The two cDNA samples retro-transcribed from total RNA derived from CS03 and CS07 cells were screened by a cDNA microarray carrying 234 human cell-cycle related genes and 1011 human signal transduction and membrane receptor-associated genes, scanned with a ScanArray 3000 laser scanner. Results The cDNA microarray analysis showed that 12 genes in CS03 were up-regulated compared to CS07, and 24 genes in CS07 were up-regulated. The function of a number of differentially expressed genes was consistently associated with cell-cycle, cell proliferation, migration, apoptosis, signal transduction and tumor metastasis, including p34 cdc2 , TSC22, plasminogen activator inhibitor I (PAI-1)and desmosome associated protein(Pinin). Conclusion Multiple genes are differentially expressed in uterine cervical carcinoma cell lines even came from the same patient. It is suggested that these genes are involved in the different phenotypic characteristics and development of cervical carcinoma.

Different responses of HepG2 subclones to low dose ethaselen

As a synthesized antineoplastic organoselenium compound, ethaselen is known to induce apoptosis in tumor cells via dose-dependent thioredoxin reductase （TrxR） inhibition. Thioredoxin, the multifunctional biological substrate of TrxR, is then left in the oxidized state, which subsequently leads to intracellular accumulation of reactive oxygen species （ROS）, cell cycle arrest and/or apoptosis. However, the low dose effect of ethaselen remains largely unknown. Several subclones have been derived from HepG2 cells by using single cell or colony isolation. The low dose of ethaselen was defined as the drug concentration of retaining 〉90% HepG2 cells alive. The HepG2 cells were used as reference of its subclones （SM01, SM02 and SM03）, and the cell cycle transition, intracellular proteins change, colony formation and sphere growth were assayed in treatment of low dose ethaselen. HepG2 and its subclones differently responded to lethal dose of cisplatin or 5-fluorouracil. Low dose of ethaselen （1 μm） modulated the cell cycle transition at 12 h of treatment, but ceils were partially recovered at 24 h of treatment though some proteins were still affected. Low dose of ethaselen did not inhibit the small colony （diameter 〉 100 μm） formation and sphere growth of HepG2 and SM01. However, low dose of ethaselen could specifically inhibit the survival, large colony （diameter 〉500 μm） formation and sphere growth of SM03, although SM03 could be rapidly recovered from ethaselen-induced cell cycle check. HepG2 and its subclone cells could survive but respond differently to treatment of low dose ethaselen （1 μM）. Low dose of ethaselen could significantly inhibit a HepG2 subclone （SM03） in cell survival and colony growth.

杜仲含药血清对成骨细胞的影响

目的:研究不同浓度杜仲含药血清对体外培养小鼠MC3T3-E1 Subclone 14成骨细胞增殖、分化的影响。方法:大鼠灌胃给予杜仲后制备含药血清,取MC3T3-E1 Subclone 14成骨细胞,分别加入空白血清(空白对照组)和含2%、5%、10%的杜仲含药血清(给药组)的培养基培养,用MTT法检测MC3T3-E1 Subclone 14成骨细胞增殖情况,ELISA法检测细胞中的碱性磷酸酶(ALP)、骨钙蛋白(OTC)、破骨细胞抑制因子与核因子κB受体活化因子配体(OPG/RANKL)系统活性。结果:与空白对照组同浓度比较,杜仲含药血清各浓度明显促进细胞增殖并上调ALP、OTC、OPG/RANKL的比值(P<0.05,P<0.01)。结论:杜仲能促进体外培养的MC3T3-E1Subclone 14成骨细胞增殖与分化。