Differential effects of mesenchymal stem cells on a heterogeneous cell population within lung cancer cell lines

Although mesenchymal stem cells (MSCs) promote lung cancer growth in vivo, in vitro studies indicate that they inhibit the proliferation of lung cancer cells. Because malignant tumors contain a heterogeneous cell population with variable capacity for self-renewal, the aim of this study was to determine whether the inconsistencies between in vitro and in vivo studies are a result of differential effects of MSCs on the heterogeneous cell population within lung cancer cell lines. Human MSCs were isolated from the bone marrow, and their cell surface antigen expression and multi-lineage differentiation capacity was examined at passage 10. CD133+ cells were isolated from A549 and H446 cell lines using immunomagnetic separation. The effects of MSCs on the growth and microsphere formation of heterogeneous cell populations within two lung cancer cell lines (A549 and H446) were compared. MSCs inhibited the in vitro proliferation of both cell lines, but significantly accelerated tumor formation and stimulated tumor growth in vivo (P < 0.05). In CD133+ cells isolated from both A549 and H446 cells, co-culture with MSCs for 1–3 days significantly increased their proliferation (P < 0.05). MSCs also significantly increased microsphere formation in both cell lines (P < 0.05). Selective stimulation of CD133+ cell growth may account for the discrepant effects of MSCs on lung cancer progression.     

The impact of C-MYC gene expression on gastric cancer cell

The upregulation or mutation of C-MYC has been observed in gastric, colon, breast, and lung tumors and in Burkitt’s lymphoma. However, little is known about the role C-MYC plays in gastric adenocarcinoma. In the present study, we intended to investigate the influence of C-MYC on the growth, proliferation, apoptosis, invasion, and cell cycle of the gastric cancer cell line SGC7901 and the gastric cell line HFE145. C-MYC cDNA was subcloned into a constitutive vector PCDNA3.1 followed by transfection in normal gastric cell line HFE145 by using liposome. Then stable transfectants were selected and appraised. Specific inhibition of C-MYC was achieved using a vector-based siRNA system which was transfected in gastric cancer cell line SGC7901. The apoptosis and cell cycles of these clones were analyzed by using flow cytometric assay. The growth and proliferation were analyzed by cell growth curves and colony-forming assay, respectively. The invasion of these clones was analyzed by using cell migration assay. The C-MYC stable expression clones (HFE-Myc) and C-MYC RNAi cells (SGC-MR) were detected and compared with their control groups, respectively. HFE-Myc grew faster than HFE145 and HFE-PC (HFE145 transfected with PCDNA3.1 vector). SGC-MR1, 2 grew slower than SGC7901 and SGC-MS1, 2 (SGC7901 transfected with scrambled control duplexes). The cell counts of HFE-Myc in the third, fourth, fifth, sixth, and seventh days were significantly more than those of control groups (P < 0.05). Those of SGC-MR1, 2 in the fourth, fifth, sixth, and seventh days were significantly fewer than those of control groups (P < 0.05). Cell cycle analysis showed that proportions of HFE-Myc and SGC-MR cells in G0–G1 and G2–M were different significantly with their control groups, respectively (P < 0.05). The apoptosis rate of HFE-Myc was significantly higher than those of control groups (P < 0.05). Results of colony-forming assay showed that the colony formation rate of HFE-Myc was higher than those of control groups; otherwise, the rate of SGC-MR was lower than those of their control groups (P < 0.05). The results of cell migration assay showed that there were no significant differences between experimental groups and control groups (P > 0.05). In conclusion, C-MYC can promote the growth and proliferation of normal gastric cells, and knockdown of C-MYC can restrain the growth and proliferation of gastric cancer cells. It can induce cell apoptosis and help tumor cell maintain malignant phenotype. But it can have not a detectable influence on the ability of invasion of gastric cancer cells.     

A PKC-β inhibitor protects against cardiac microvascular ischemia reperfusion injury in diabetic rats

PKC-β inhibitor Ruboxistaurin (RBX or LY333531) can be used to reverse diabetic microvascular complication. However, it has not been previously established whether RBX can protect against ischemia/reperfusion (I/R) injury of cardiac microvessels in diabetic rats. STZ-induced diabetic rats were randomized into four groups and underwent I/R procedures. Cardiac barrier function and the region of cardiac microvascular lesion were examined. Cell monolayer barrier function was detected in cultured cardiac microvascular endothelial cells (CMECs) subjected to simulated I/R (SI/R). PKC-β siRNA was transfected into CMECs to silence PKC-β. Apoptosis Index of CMECs was detected by TUNEL assay and phosphor-LIMK2 protein expression was examined by Western blot analysis. RBX and insulin administration significantly reduced the cardiac microvascular lesion region and Apoptosis Index of endothelial cells (all P < 0.05 vs. no-treatment group). RBX decreased phosphor-LIMK2 expression (P < 0.05 vs. no-treatment group). RBX pretreatment and transfection with PKC-β siRNA induced a rapid barrier enhancement in CMECs monolayer as detected by increased transendothelial electrical resistance (TER) and decreased FITC-dextran clearance (all P < 0.05 vs. no-treatment group). Meanwhile, RBX pretreatment and transfection with PKC-β siRNA significantly decreased TUNEL positive CMECs and phosphor-LIMK2 expression in cultured CMECs (all P < 0.05 vs. no-treatment group). RBX pretreatment reduced F-actin/G-actin in cultured CMECs, reproducing the same effect as PKC-β siRNA. These data indicate that PKC-β inhibitor (RBX) may be helpful in attenuating the risk of severe cardiac microvascular I/R injury in diabetic rats partly due to its maintenance of endothelial barrier function and anti-apoptotic effect.     

Effect of ARHI on lung cancer cell proliferation, apoptosis and invasion in vitro

The purposes of this study were to elucidate the effects of ARHI (aplysia ras homolog I) on several biological features of lung cancer cells, including growth, proliferation and invasion, to collect experimental evidence for the future biological treatment of human lung cancer. The eukaryotic expression vector, pcDNA3.1–ARHI, was constructed and transfected into the human lung cancer cell line SK-MES-1. The biological properties of the resulting ARHI-expressing lung cancer cell line were evaluated using methyl thiazolyl tetrazolium assay, flow cytometry, and a Transwell invasion assay. Additionally, the influence of ARHI on the gene expression levels of cyclin D1, p27^KIP1, death-associated protein kinase 1 (DAPK1), and matrix metalloproteinases1/2 (MMP-1/2) was determined. Compared to the non-transfected SK-MES-1 cells and the cells transfected with the empty pcDNA3.1 plasmid, the ARHI-transfected cells displayed significantly reduced growth rates and decreased viability (P < 0.05). The ARHI-transfected cells also displayed a significantly higher percentage of cells in G1 phase (P < 0.05) and a lower percentage of cells in S phase (P < 0.05); a higher percentage of apoptosis (P < 0.05); and finally, a notable reduction in the basement membrane-penetration rate in the Transwell invasion assay (P < 0.05). Furthermore, it was determined that ARHI is capable of inhibiting the expression of cyclin D1, MMP-1, and MMP-2; however, ARHI promotes the expression of both p27^KIP1 and DAPK1 in SK-MES-1 cells. In conclusion, overexpression of ARHI gene might be associated with the inhibition of lung cancer cell growth, proliferation and invasion, and the promotion of apoptosis.     

The effects on cell growth and chemosensitivity by livin RNAi in non-small cell lung cancer

Livin is highly expressed in most tumor tissues and could inhibit the tumor cells apoptosis. Knockdown of endogenous livin expression in non-small cell lung cancer (NSCLC) cells could inhibit cell growth. But it is still unclear if knockdown of endogenous livin expression combined with conventional chemotherapy could play a positive role in NSCLC treatment. In this article, the efficient RNA interferences (RNAi) of livin were constructed, and then we transfected them into A549 cells and 103H cells to study their influence on cell cycle and apoptosis index. At last, we detected the cell's sensitivity to conventional chemotherapeutic drugs after knockdown endogenous livin expression in A549 cells and 103H cells. Our results showed that knockdown livin expression could inhibit cell growth and induce apoptosis in A549 cells and 103H cells. A549 cells and 103H cells had an increased chemosensitivity to adriamycin and cisplatin after transfection of livin RNAi constructs. The results indicated that cell cycle redistribution and increased apoptosis index after knockdown livin expression might provide the main explanation for the enhanced chemosensitivity. Proper combination of livin RNAi and some conventional chemotherapeutic drugs may entail potential benefits in the treatment of NSCLC.     

Regulation of retinal endothelial cell apoptosis through activation of the IGFBP-3 receptor

The goal of this study was to investigate whether insulin-like growth factor binding protein-3 receptor (IGFBP-3 receptor) is required for IGFBP-3 to inhibit retinal endothelial cell (REC) apoptosis. REC were grown in normal glucose (5 mM) or high glucose medium (25 mM) for 3 days. Once cells reached confluence, they were transfected with an endothelial- specific IGFBP-3 plasmid DNA (non-IGF binding; IGFBP-3 NB) at 1 μg/ml for 24 h. Cell proteins were extracted and analyzed for IGFBP-3 receptor expression by Western blotting or use in coimmunoprecipitation or co-localization experiments for detection of IGFBP-3 and IGFBP-3 receptor binding. REC were also transfected with or without IGFBP-3 receptor siRNA before IGFBP-3NB plasmid DNA transfection. Cell lysates were processed for a cell death ELISA, a cleaved caspase 3 ELISA, and Western blotting to measure key pro- and anti-apoptotic markers: Bcl-xL, Bax, Cytochrome C and Akt. The IGFBP-3 receptor is present on REC. Overexpression of IGFBP-3 in REC significantly increased protein levels of IGFBP-3 receptor (p < 0.05). Significant increases in cell death were found in cells transfected with IGFBP-3 receptor siRNA versus not treated samples (p < 0.05). Data suggest that IGFBP-3 inhibits retinal endothelial cell death through activation of an IGFBP-3 receptor in a hyperglycemic environment. This is the first demonstration of the involvement of IGFBP-3 receptor in inhibition of REC cell death. Future studies will investigate the mechanism by which IGFBP-3 receptor may inhibit retinal endothelial cell death.     

Lentiviral vector-mediate ATG3 overexpression inhibits growth and promotes apoptosis of human SKM-1 cells

Based on the nested case–control study cohort and gene expression profile, we have picked up a subset of six genes to distinguish the leukemia group and control group stably. ATG3 is the only down regulated gene. This research is to investigate the effect of ATG3 gene over expression by lentivirus on SKM-1 cell line and myelodysplastic syndrome to leukemic transformation. Human SKM-1 cells were transfected with ATG3–GFP recombinant lentiviral vectors and compared with cells transfected with GFP lentiviral vectors. Western blot was performed to detect the ATG3 protein. Cell proliferation was assessed by cell counting kit-8. Cell vitality was tested by Trypan Blue. Cell apoptosis was determined by Annexin V Apoptosis Detection Kit APC. Observe and compare the changes on growth curve, cell vitality and cell apoptosis. After 72 h of transfection, satisfactory transfection efficiency (> 90 %) was observed. SKM-1 cell line showed a statistically significant (P < 0.05) overexpression of ATG3, parallel to significantly (P < 0.05) inhibited cell proliferation. The cell vitality of ATG3 overexpression was significantly (P < 0.05) lower than negative control. Cell apoptosis analysis by flow cytometer demonstrated decreased proportion of early apoptosis and increased that of late apoptosis and death (P < 0.05). Over expressed ATG3 gene and protein, the SKM-1 cell line was inhibited in proliferation and cell vitality. It was promoted from early apoptosis to late apoptosis and death. The malignancy of SKM-1 cell line was decreased after transfection. ATG3 gene and its gene family may play an important role in transformation of myelodysplastic syndrome.     

The Role of NQO1 Polymorphisms in the Susceptibility and Chemotherapy Response of Chinese NSCLC Patients

To investigate whether the NAD(P)H:quinone oxidoreductase 1 (NQO1) gene polymorphisms determine the Platinum-based chemotherapy in advanced non-small cell lung cancer (NSCLC) in a Chinese cohort. A total of 391 patients with inoperable advanced stage of NSCLC, namely, stage III (A + B) and IV NSCLC, and 663 age-and sex-matched healthy were enrolled. The effects of chemotherapy were evaluated. NQO1 C609T polymorphism was determined. The NSCLC patients had a significantly higher prevalence of TT than control subjects (33.76 vs. 21.67 %, P < 0.001). For allele comparison, NSCLC subjects had lower T allele frequency than controls as well (55.63 vs. 44.42 %, P < 0.001). multivariate regression analyses showed the TT carriage had a significantly increased risk for development of NSCLC after adjustments with age, sex, smoke, and cancer family history (OR 1.681, 95 % CI 1.242–2.274, P = 0.001). The TT genotype distribution was significantly higher in non-responders than in responders (31.85 vs. 21.96 %, P = 0.003). Logistic regression analysis showed TT genotype carriers had less chance to gain chemotherapy response compared to CC genotype carriers (OR 0.399, P = 0.003) after adjustment with sex, age, tumor histology, disease stage, and chemotherapy regimens. The NQO1 C609T polymorphism is an important molecular marker for advanced NSCLC, since it is associated with the NSCLC risk as well as the response status of platinum-based chemotherapy.     

MicroRNA-575靶向抑制BLID促进非小细胞肺癌细胞的增殖和侵袭

目的:通过体外实验探讨miR-575对非小细胞肺癌(NSCLC)细胞增殖与侵袭能力的影响及相关机制。方法:采用实时定量PCR法检测不同非小细胞肺癌细胞系中miR-575、BLID的表达;CCK-8法检测转染miR-575模拟物、抑制因子后不同时间A549细胞增殖情况的变化;Transwell法检测A549细胞的侵袭情况;Targetcan法及双荧光素酶检测miR-575对BLID 3'UTR端的靶向作用;Western blot法检测BLID蛋白的表达。结果:A549、SPC-A1、H1299、H1650等人非小细胞肺癌细胞系中miR-575的表达均显著高于永生化的人支气管上皮细胞系16HBE(P0.001)。MiR-575模拟物转染的A549细胞miR-575的表达明显高于对照组(P0.001),同时细胞的增殖和侵袭力增强(P0.05);反之,miR-575抑制因子转染的A549细胞miR-575的表达显著降低,且细胞的增殖和侵袭力明显降低(P0.01)。Targetscan法预测BLID可能是miR-575的下游靶基因,荧光素酶结果显示miR-575不仅能够有效抑制野生型BLID 3'UTR端的荧光素酶反应(P0.01),而且能够降低BLID的蛋白表达量(P0.01)。实时定量PCR结果显示BLID在NSCLC细胞系中均呈现显著的低表达(P0.001),且转染BLID后,NSCLC细胞的增殖和细胞侵袭被明显抑制(P0.05),而当miR-575与BLID共转染时,miR-575能够逆转BLID所抑制的细胞增殖和侵袭(P0.01)。结论:在NSCLC细胞系中,miR-575的表达上调,且能够通过直接作用于下游靶点抑癌基因BLID从而促非小细胞肺癌细胞增殖及侵袭。     

MiR‐376a suppresses the proliferation and invasion of non‐small‐cell lung cancer by targeting c‐Myc

It has been reported that miR‐376a is involved in the formation and progression of several types of cancer. However, the expression and function of miR‐376a is still unknown in non‐small cell lung carcinomas (NSCLC). In this study, the expression of miR‐376a in NSCLC tissues and cell lines were examined by real‐time PCR, the effects of miR‐376a on cell proliferation, apoptosis and invasion were evaluated in vitro. Luciferase reporter assay was performed to identify the targets of miR‐376a. The results showed that miR‐376a was significantly downregulated in NSCLC tissues and cell lines. Restoration of miR‐376a in NSCLC cell line A549 significantly inhibited cell proliferation, increased cell apoptosis and suppressed cell invasion, compared with control‐transfected A549 cells. Luciferase reporter assay showed that c‐Myc, an oncogene that regulating cell survival, angiogenesis and metastasis, was a direct target of miR‐376a. Over‐expression of miR‐376a decreased the mRNA and protein levels of c‐Myc in A549 cells. In addition, upregulation of c‐Myc inhibited miR‐376a‐induced inhibition of cell proliferation and invasion in A549 cells. Therefore, our results indicate a tumor suppressor role of miR‐376a in NSCLC by targeting c‐Myc. miR‐376a may be a promising therapeutic target for NSCLC.     

Screening and test of CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector

This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P < 0.01) change in expression levels were screened according to the screening standard (signal log ratio ≥1 or ≤?1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335 ± 0.0116 72 h after transfection. This value was significantly (P < 0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P < 0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.     

Bcl-2 small hairpin RNAs enhance radiation-induced apoptosis in A549 cells

Bcl-2, a prominent member of the family of proteins, is responsible for dys-regulation of apoptosis and resistance to chemotherapy and radiotherapy. This study investigated whether small hairpin RNA (shRNA) targeting Bcl-2 could render A549 cells more susceptible to gamma radiation-induced apoptosis. Recombinant Bcl-2 shRNAs expression vector were transfected into A549 cells with Lipofectamine 2000. Transfected cells were screened in 800 mg/ml G418 screening medium, and after stable transfection, silencing was examined. Expression of the Bcl-2 protein was assayed using Western blot in A549 cells. Inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by morphological observation and flow cytometry. Expression levels of Bcl-2 protein from A549 cells decreased after stable transfection with Bcl-2 shRNAs. No differences in Bcl-2 protein levels between control shRNA group and untreated cells were noted. After stable transfection with Bcl-2 shRNAs the viability of cells was less than after stable transfection with those with control shRNAs and untransfected A549, respectively (P<0.05). Control shRNA had no significant effect on growth of cells. Radiation significantly inhibited the growth of cells stably transfected with Bcl-2 shRNA (P<0.05). No difference in survival between the cells with control shRNA and untransfected cells was noted. Using Giemsa staining, cells stably transfected with Bcl-2 shRNA combined with radiation at 48 h displayed changes of apoptosis. After treatment with radiation apoptotic rates of the A549 cells stably transfected with Bcl-2 shRNA significantly increased (P<0.05), compared with the cells with control shRNA and untransfected cells. shRNAs against the Bcl-2 mRNA increases radiation-induced apoptosis in A549 cells.     

Neuropilin 1 expression correlates with the Radio‐resistance of human non‐small‐cell lung cancer cells

The purpose of this study was to determine the correlation between over‐expression of the neuropilin 1 (NRP1) gene and growth, survival, and radio‐sensitivity of non‐small cell lung carcinoma (NSCLC) cells. 3‐[4,5‐dimethylthylthiazol‐2‐yl]‐2,5 diphenyltetrazolium broide (MTT) and colony assays were then performed to determine the effect of NRP1 inhibition on the in vitro growth of NSCLC cells. The Annexin V‐Fluorescein Isothiocyanate (FITC) apoptosis detection assay was performed to analyse the effect of NRP1 enhancement on apoptosis of NSCLC cells. Transwell invasion and migration assays were employed to examine the metastatic ability of A549 cells post X‐ray irradiation. In addition, Western blot assays were carried out to detect the protein level of VEGFR2, PI3K and NF‐κB. Finally, to examine the effect of shNRP1 on proliferation and radio‐sensitivity in vivo, a subcutaneous tumour formation assay in nude mice was performed. Microvessel density in tumour tissues was assessed by immunohistochemistry. The stable transfected cell line (shNRP1‐A549) showed a significant reduction in colony‐forming ability and proliferation not only in vitro, but also in vivo. Moreover, shRNA‐mediated NRP1 inhibition also significantly enhanced the radio‐sensitivity of NSCLC cells both in vitro and in vivo. The over‐expression of NRP1 was correlated with growth, survival and radio‐resistance of NSCLC cells via the VEGF‐PI3K‐ NF‐κB pathway, and NRP1 may be a molecular therapeutic target for gene therapy or radio‐sensitization of NSCLC.     

The Thr300Ala variant of ATG16L1 is associated with decreased risk of brain metastasis in patients with non-small cell lung cancer

Non-small cell lung cancer (NSCLC) often metastasizes to the brain, but identifying which patients will develop brain metastases (BM) is difficult. Macroautophagy/autophagy is critical for cancer initiation and progression. We hypothesized that genetic variants of autophagy-related genes may affect brain metastases (BM) in NSCLC patients. We genotyped 16 single nucleotide polymorphisms (SNPs) in 7 autophagy-related (ATG) genes (ATG3, ATG5, ATG7, ATG10, ATG12, ATG16L1, and MAP1LC3/LC3) by using DNA from blood samples of 323 NSCLC patients. Further, we evaluated the potential associations of these genes with subsequent BM development. Lung cancer cell lines stably transfected with ATG16L1: rs2241880 (T300A) were established. Mouse models of brain metastasis were developed using cells transfected with ATG16L1–300T or ATG16L1–300A. ATG10: rs10036653 and ATG16L1: rs2241880 were significantly associated with a decreased risk of BM (respective hazard ratios [HRs]=0.596, 95% confidence interval [CI] 0.398–0.894, P = 0.012; and HR = 0. 655, 95% CI 0.438–0.978, P = 0.039, respectively). ATG12: rs26532 was significantly associated with an increased risk of BM (HR=1.644, 95% CI 1.049–2.576, P = 0.030). Invasion and migration assays indicated that transfection with ATG16L1–300T (vs. 300A) stimulated the migration of A549 cells. An in vivo metastasis assay revealed that transfection with ATG16L1–300T (vs. 300A) significantly increased brain metastasis. Our results indicate that genetic variations in autophagy-related genes can predict BM and that genome analysis would facilitate stratification of patients for BM prevention trials.     

Expression of periostin in the serum of NSCLC and its function on proliferation and migration of human lung adenocarcinoma cell line (A549) in vitro

Periostin is over expressed in many epithelial malignant cancers, including lung cancer, breast cancer, ovarian cancer and colon cancer. It is related with the progression and migration of breast and ovarian cancer cells in vitro. The aim of this study was to investigate the serum level of periostin in non-small cell lung cancer (NSCLC) and its relationship with established biological and prognostic factors by enzyme-linked-immunosorbent serologic assay. We also observe the function of periostin on the proliferation and migration of human lung adenocarcinoma cell line (A549) and discuss the mechanism. The mean value for serum periostin (POSTN) was elevated in NSCLC patients (242.84 ± 5.33 pg/ml) compared to the normal healthy volunteers (215.66 ± 11.67 pg/ml) (p = 0.030). The serum level of periostin of NSCLC patients had no connection with gender, age, pathological type, TNM stage, lymph node status, tumor size and invasiveness. We constructed a plasmid named pEGFP-N1/POSTN expressing full-length human periostin. Transfecting the plasmid to A549 cells and periostin was efficiently expressed in transfected A549 cells. Our data showed that periostin could promote the proliferation and migration of A549 cells by inducing vimentin and N-cadherin expression and downregulating E-cadherin expression. These results strongly suggest that periostin is a novel molecular which play an important role during the progression and development of NSCLC.     

Optimization of human umbilical cord mesenchymal stem cell isolation and culture methods

Human umbilical cord mesenchymal stem cells (hUCMSCs) are considered to be an ideal replacement for bone marrow MSCs. However, up to date, there is no convenient and efficient method for hUCMSC isolation and culture. The present study was carried out to explore the modified enzyme digestion for hUCMSC in vitro. Conventional enzyme digestion, modified enzyme digestion, and tissue explant were used on hUCMSCs to compare their efficiencies of isolation and culture, to observe primary cell growth and cell subculture. The results show that the cells cultured using the tissue explant method had a longer culture cycle (P < 0.01) and lower yield of primary cells per centimetre of umbilical cord (P < 0.01) compared with the two enzyme digestion methods. Subculture adherence and cell doubling took significantly less time with the tissue explant method (P < 0.05) than with the conventional enzyme digestion method; however, there was no significant difference between the tissue explant method and the modified enzyme digestion method (P > 0.05). Comparing two enzyme digestion methods, the modified method yielded more cells than did the conventional method (P < 0.01), and primary cell adherence took significantly less time with the modified method than with the conventional method (P < 0.05). Cell cycle analysis of the third-generation hUCMSCs cultured by modified enzyme digestion method indicated that most cells were quiescent. Immunofluorescence staining showed that these cells expressed MSC markers CD44 and CD90. And Von Kossa and oil red O staining detection showed that they could be differentiated into osteoblasts and adipocytes with induction medium in vitro. This study suggests that hUCMSC isolation and culture using 0.2 % collagenase II at 37 °C for digestion of 16–20 h is an effective and simple modified enzyme digestion method.