Microarray Study of Mechanism of Trichostatin A Inducing Apoptosis of Molt-4 Cells

Histone deacetylase was overexpressed in a variety of cancers and was closely correlated with oncogenic factors. The histone deacetylase inhibitor, trichostatin A （TSA） was shown to induce apoptosis in many cancer cells. However, the mechanism of TSA on induction of cancer cells apoptosis is poorly understood. This study was designed to characterize the global gene expression profiles before and after treatment of human leukemia cell line Molt-4 with TSA. Flow cytometry, MTT and DNA ladder were used to observe the effect of TSA on the apoptosis of MOLT-4 cells and normal human peripheral blood mononuclear cells （PBMC）. Microarray, reverse transcription-polymerase chain reaction （RTopCR） and Western blotting were used to detect the difference of gene and protein expressions of Molt-4 cells after incubation of the cells with TSA. The results showed that TSA could induce Molt-4 apoptosis in dose- and time-dependent manners but spared PBMCs. Microarray analysis showed that after incubation with TSA for 9 h, 310 genes were upregulated and 313 genes were deregulated. These genes regulate the growth, differentiation and survival of cells. Among these genes, STATSA was down-regulated by 80.4% and MYC was down-regulated by 77.3%. It was concluded that TSA has definite growth-inhibiting and apoptosis-inducing effects on Molt-4 cells in time- and dose-dependent manners, with weak cytotoxic effects on PBMCs at the same time. The mechanism of TSA selectively inducing apoptosis and inhibiting growth may be ascribed to the changes of pro-proliferation genes and anti-apoptosis genes.

Apoptosis-inducing effect of Jinke on Molt-4 cells and its mechanism

Objective： To investigate the apoptosis-inducing effect of Jinke on Molt-4 cells and its possible mechanism. Methods： The Molt-4 cells were treated with different concentrations of Jinke and then cultured for necessary time. The Annexin-V / PI method was used to detect the apoptosis rate. The cell cycle was analyzed by DNA content with flow cytometry. Double parameters analysis of cyclins / DNA was performed to detect the expression of cyclin E. API method was used to confirm the cell cycle-specific apoptosis. The expressions of Bcl-2 and Bax were detected by westem blot. Results： 24 h after the treatment of 0.5, 1.0, 1.5, 2.0 and 3.0 mg/mL Jinke, the apoptosis rate of Molt-4 cells was evaluated in a concentration-dependent manner, from 5.2% of the control group to 41.0% of the 3.0 mg/mL Jinke group. When the Molt-4 cells were cultured with 1.5 mg/mL Jinke, the apoptosis rate was evaluated in a time-dependent manner. DNA content analysis showed that G0/G1 phase of Molt-4 cells increased in a time-dependent manner. The expression of cyclin E increased gradually. API assay showed the apoptosis cells were almost in G0/G1 phase. Western blot showed the Bcl-2 was down-regulated and the Bax was up-regulated. Conclusion： Jinke could induce G1 phase-specific apoptosis in Molt-4 cells in time-and concentration-dependent manners involving G1 phase arrest. The mechanism of apoptosis inducing effect may be related to the upregulation of Bax and the down-regulation of Bcl-2.

Effects of Trichostatin A on HDAC8 Expression,Proliferation and Cell Cycle of Molt-4 Cells

The effects of Trichostatin A （TSA） on histone deacetylase 8 （HDAC8） expression, proliferation and cell cycle arrest in T-lymphoblastic leukemia cell line Molt-4 cells in vitro were investigated. The effect of TSA on the growth of Molt-4 cells was studied by MTT assay. Flow cytometry was used to examine the cell cycle. The expression of HDAC8 was detected by using immunocytochemistry and Western blot. The results showed that proliferation of Molt-4 cells was inhibited in TSA-treated group in a time- and dose-dependent manner, The IC50 of TSA exposures for 24 h and 36 h were 254.3236 and 199.257 μg/L respectively. The cell cycle analysis revealed that Molt-4 was mostly in G0/G1 phase, and after treatment with TSA from 50 to 400 μg/L for 24 h, the percents of G0/G1 cells were decreased and cells were arrested in GJM phase. Treatment of TSA for 24 h could significantly inhibit the expression of HDAC8 protein in Molt-4 cells （P〈0.01）. It was concluded that TSA could decrease the expression of HDAC8 in Molt-4 cells, which contributed to the inhibition of proliferation and induction of cell cycle arrest in Molt-4 cells.

Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

Microglial activation that occurs rapidly after closed head injury may play important and complex roles in neuroinflammation-associated neuronal damage and repair.We previously reported that induced neural stem cells can modulate the behavior of activated microglia via CXCL12/CXCR4 signaling,influencing their activation such that they can promote neurological recovery.However,the mechanism of CXCR4 upregulation in induced neural stem cells remains unclear.In this study,we found that nuclear factor-κB activation induced by closed head injury mouse serum in microglia promoted CXCL12 and tumor necrosis factor-αexpression but suppressed insulin-like growth factor-1 expression.However,recombinant complement receptor 2-conjugated Crry(CR2-Crry)reduced the effects of closed head injury mouse serum-induced nuclear factor-κB activation in microglia and the levels of activated microglia,CXCL12,and tumor necrosis factor-α.Additionally,we observed that,in response to stimulation(including stimulation by CXCL12 secreted by activated microglia),CXCR4 and Crry levels can be upregulated in induced neural stem cells via the interplay among CXCL12/CXCR4,Crry,and Akt signaling to modulate microglial activation.In agreement with these in vitro experimental results,we found that Akt activation enhanced the immunoregulatory effects of induced neural stem cell grafts on microglial activation,leading to the promotion of neurological recovery via insulin-like growth factor-1 secretion and the neuroprotective effects of induced neural stem cell grafts through CXCR4 and Crry upregulation in the injured cortices of closed head injury mice.Notably,these beneficial effects of Akt activation in induced neural stem cells were positively correlated with the therapeutic effects of induced neural stem cells on neuronal injury,cerebral edema,and neurological disorders post–closed head injury.In conclusion,our findings reveal that Akt activation may enhance the immunoregulatory effects of induced neural stem cells on microglial activation via upregulation of CXCR4 and Crry,thereby promoting induced neural stem cell–mediated improvement of neuronal injury,cerebral edema,and neurological disorders following closed head injury.

Targeting STAT3 with SH-4-54 suppresses stemness and chemoresistance in cancer stem-like cells derived from colorectal cancer

BACKGROUND Over the years,the numbers of treatment options for colorectal cancer(CRC)have increased,leading to notable improvements in the overall survival of CRC patients.Although therapy may initially yield positive results,the development of drug resistance can result in treatment failure and cancer recurrence.This resistance is often attributed to the presence of cancer stem cells(CSCs).These CSCs not only contribute to therapeutic resistance but also play crucial roles in the initiation and development of tumor metastasis.AIM To investigate the antitumor effects of SH-4-54,which are mediated by targeting CSCs relative to treatment outcomes.METHODS CSCs were enriched by culturing CRC cells in serum-free medium.Hallmarks of stemness and IL-6/JAK2/STAT3 signaling were detected by Western blotting.Indicators of CSC malignancy,including proliferation,invasion,and tumor formation,were measured.RESULTS In this study,we employed SH-4-54,which exhibits anticancer activity in solid tumors through targeting the SH2 domain of both the signal transducer and activator of transcription(STAT)3 and the STAT5,and evaluated its effects on stemness and chemoresistance in colorectal CSCs.As expected,SH-4-54 treatment inhibited the phosphorylation of STAT3(p-STAT3)and decreased the percentage of ALDH1A1-positive CRC cells.The addition of SH-4-54 dissociated colorectal spheroids and decreased the expression of stemness markers,including ALDH1A1,CD44 and Nanog.SH-4-54 treatment decreased IL-6/JAK2/STAT3 signaling by inhibiting p-STAT3 and thus inhibited spheroid formation by SW480 and LoVo cells.Moreover,SH-4-54 treatment inhibited indicators of malignancy,including cell proliferation,invasion,and tumor formation,in CSCs in vitro and in vivo.Notably,SH-4-54 treatment significantly increased chemosensitivity to oxaplatin.CONCLUSION Taken together,these results indicate that SH-4-54 is a promising molecule that exerts antitumor effects on colorectal CSCs by inhibiting STAT3 signaling.

Role of octamer transcription factor 4 in proliferation,migration,drug sensitivity,and stemness maintenance of pancreatic cancer cells

BACKGROUND Pancreatic cancer(PC)is one of the most aggressive malignancies characterized by rapid progression and poor prognosis.The involvement of cancer stem cells(CSCs)and Octamer transcription factor 4(OCT4)in PC pathobiology is being increasingly recognized.AIM To investigate the role of OCT4 in pancreatic CSCs and its effect on PC cell prolif-eration,migration,drug sensitivity,and stemness maintenance.METHODS We analyzed OCT4 and CD133 expression in PC tissues and cell lines.BxPC-3 cells were used to assess the effects of OCT4 modulation on cellular behavior.Proliferation,migration,and stemness of BxPC-3 cells were evaluated,and the PI3K/AKT/mTOR pathway was examined to gain mechanistic insights.RESULTS OCT4 and CD133 were significantly overexpressed in PC tissues.OCT4 mo-dulation altered BxPC-3 cell proliferation,invasion,and stemness,with OCT4 overexpression(OV-OCT4)enhancing these properties and OCT4 interference decreasing them.OV-OCT4 activated the PI3K/AKT/mTOR pathway,which correlated with an increase in PC stem cells(PCSC).CONCLUSION OCT4 plays a crucial role in PCSCs by influencing the aggressiveness and drug resistance of PC cells,thus presenting itself as a potential therapeutic target.

Small molecule inhibitor DDQ-treated hippocampal neuronal cells show improved neurite outgrowth and synaptic branching

The process of neurite outgrowth and branching is a crucial aspect of neuronal development and regeneration.Axons and dendrites,sometimes referred to as neurites,are extensions of a neuron's cellular body that are used to start networks.Here we explored the effects of diethyl(3,4-dihydroxyphenethylamino)(quinolin-4-yl)methylphosphonate(DDQ)on neurite developmental features in HT22 neuronal cells.In this work,we examined the protective effects of DDQ on neuronal processes and synaptic outgrowth in differentiated HT22cells expressing mutant Tau(mTau)cDNA.To investigate DDQ chara cteristics,cell viability,biochemical,molecular,western blotting,and immunocytochemistry were used.Neurite outgrowth is evaluated through the segmentation and measurement of neural processes.These neural processes can be seen and measured with a fluorescence microscope by manually tracing and measuring the length of the neurite growth.These neuronal processes can be observed and quantified with a fluorescent microscope by manually tracing and measuring the length of the neuronal HT22.DDQ-treated mTau-HT22 cells(HT22 cells transfected with cDNA mutant Tau)were seen to display increased levels of synaptophysin,MAP-2,andβ-tubulin.Additionally,we confirmed and noted reduced levels of both total and p-Tau,as well as elevated levels of microtubule-associated protein 2,β-tubulin,synaptophysin,vesicular acetylcholine transporter,and the mitochondrial biogenesis protein-pe roxisome prolife rator-activated receptor-gamma coactivator-1α.In mTa u-expressed HT22 neurons,we observed DDQ enhanced the neurite characteristics and improved neurite development through increased synaptic outgrowth.Our findings conclude that mTa u-HT22(Alzheimer's disease)cells treated with DDQ have functional neurite developmental chara cteristics.The key finding is that,in mTa u-HT22 cells,DDQ preserves neuronal structure and may even enhance nerve development function with mTa u inhibition.

Label-free in-vivo classi-cation and tracking of red blood cells and platelets using Dynamic-YOLOv4 network

In-vivo flow cytometry is a noninvasive real-time diagnostic technique that facilitates continuous monitoring of cells without perturbing their natural biological environment,which renders it a valuable tool for both scientific research and clinical applications.However,the conventional approach for improving classification accuracy often involves labeling cells with fluorescence,which can lead to potential phototoxicity.This study proposes a label-free in-vivo flow cytometry technique,called dynamic YOLOv4(D-YOLOv4),which improves classification accuracy by integrating absorption intensity fluctuation modulation(AIFM)into YOLOv4 to demodulate the temporal features of moving red blood cells(RBCs)and platelets.Using zebrafish as an experimental model,the D-YOLOv4 method achieved average precisions(APs)of 0.90 for RBCs and 0.64 for thrombocytes(similar to platelets in mammals),resulting in an overall AP of 0.77.These scores notably surpass those attained by alternative network models,thereby demonstrating that the combination of physical models with neural networks provides an innovative approach toward developing label-free in-vivoflow cytometry,which holds promise for diverse in-vivo cell classification applications.

Enhancing the crystallinity and stability of perovskite solar cells with 4-tert-butylpyridine induction for efficiency exceeding 24%

Perovskite solar cells(PSCs)have emerged as a promising photovoltaic technology because of their high light absorption coefficient,long carrier diffusion distance,and tunable bandgap.However,PSCs face challenges such as hysteresis effects and stability issues.In this study,we introduced a novel approach to improve film crystallization by leveraging 4-tert-butylpyridine(TBP)molecules,thereby enhancing the performance and stability of PSCs.Our findings demonstrate the effective removal of PbI_(2)from the perovskite surface through strong coordination with TBP molecules.Additionally,by carefully adjusting the concentration of the TBP solution,we achieved enhanced film crystallinity without disrupting the perovskite structure.The TBP-treated perovskite films exhibit a low defect density,improved crystallinity,and improved carrier lifetime.As a result,the PSCs manufactured with TBP treatment achieve power conversion efficiency(PCE)exceeding 24%.Moreover,we obtained the PCE of 21.39%for the 12.25 cm^(2)module.

Protective effect of erythropoietin against 1-methyl-4-phenylpyridinium-induced neurodegenaration in PC12 cells

Objective The neuroprotective effect of erythropoietin （EPO） against 1-methyl-4-phenylpyridinium （MPP^＋）- induced oxidative stress in cultured PC12 cells, as well as the underlying mechanism, were investigated. Methods PC12 ceils impaired by MPP^＋ were used as the cell model of Parkinson＇s disease. Methyl thiazolyl tetrazolium （MTT） was used to assay the viability of the PC12 cells exposed to gradient concentrations of EPO, and the terminal deoxynucleotidyl transferase dUTP nick end labelling （TUNEL） assay was used to analyze the apoptosis ratio of PC 12 cells. The expression of Bcl-2 and Bax in PC 12 cells were examined by Western blot, and the reactive oxygen species （ROS）, the mitochondrial transmembrane potential and the activity of caspase-3 in each group were detected by spectrofluorometer. Results Treatment of PC12 cells with MPP^＋ caused the loss of cell viability, which may be associated with the elevation in apoptotic rate, the formation of ROS and the disruption of mitochondrial transmembrane potential. It was also shown that MPP＋ significantly induced the upregulation of Bax/Bcl-2 ratio and the activation of caspase-3. In contrast, EPO significantly reversed these responses and had the maximum protective effect at 1 U/mL. Conclusion The inhibitive effect of EPO on the MPP^＋ -induced cytotoxicity may be ascribed to its anti-oxidative property and anti-apoptotic activity, and EPO may provide a useful therapeutic strategy for treatment of neurodegenerative diseases such as Parkinson＇s disease.

Dual effect of Molt-4 cell line onset with roscovitine

Objective： To observe the effect of roscovitine （ROSC） as some Cyclin-dependent kinases （CDKs）, inhibitor onset with the Molt-4 cell lines. Methods： DNA assay of single cells by flow cytometry was used to detect the effect of cell cycle arrest and Annexin-V/FITC assay was used to detect the effect of cell apoptosis when the Molt-4 cell lines cultured with different concentration ROSC in different time points. Results： It showed that ROSC exerted strong inhibitory effect on proliferation and cell cycle progression of Molt-4 cell lines, accumulation of G2/M cells arrested took place after onset with 10 IJm and 20 μm ROSC more than 6 h; at the same time, the cell apoptosis of Molt-4 cells would be detected. According with the time and concentration increasing, the cell apoptosis rate would rise. Conclusion： It is concluded that Roscovitine （ROSC） as some Cyclin-dependent kinases （CDKs）, inhibitor, has dual effects to Molt-4 cells, not only the effect of cell cycle arrest but also the effect of cell apoptosis.

血管生成素样蛋白4通过调节成纤维细胞和内皮细胞功能影响糖尿病足进程

背景:研究表明,血管因素对糖尿病足的发生具有重要影响。目的:探讨血管生成素样蛋白4在糖尿病足形成中的重要作用。方法:①对糖尿病足患者的基因表达谱数据进行生物信息学分析,找到关键基因。收集糖尿病足患者以及无糖尿病健康人的皮肤标本进行苏木精-伊红染色、免疫组化染色以及qRT-PCR实验,检测血管生成素样蛋白4表达情况。②培养人永生化皮肤成纤维细胞系和原代人脐静脉内皮细胞,将2种细胞分别分为对照组和外源性补充血管生成素样蛋白4组,通过划痕实验以及CCK-8实验分别检测成纤维细胞的迁移能力和增殖能力,通过Ki67实验检测内皮细胞的增殖能力。结果与结论:①生信分析发现,血管生成素样蛋白4基因的下调可能是导致糖尿病足形成的关键基因。②苏木精-伊红染色结果显示,与正常皮肤相比,血管生成素样蛋白在糖尿病足皮肤内弱表达,且其mRNA水平相对表达量降低(P<0.01)。③划痕实验结果显示,与对照组相比,血管生成素样蛋白4组成纤维细胞迁移能力明显增强;CCK-8细胞增殖实验显示,血管生成素样蛋白4组成纤维细胞的吸光度值在24,48 h均高于对照组(P<0.01,P<0.001);提示血管生成素样蛋白4可增强高糖处理的成纤维细胞迁移及增殖能力。④Ki67实验结果显示,与对照组相比,血管生成素样蛋白4组内皮细胞Ki67阳性细胞数目明显多于对照组;CCK-8细胞增殖实验显示,血管生成素样蛋白4组内皮细胞的吸光度值在24,48 h均高于对照组(P<0.05,P<0.001)。(5)以上结果均提示血管生成素样蛋白4可增强高糖处理内皮细胞的增殖能力。

Chemokine platelet factor 4 accelerates peripheral nerve regeneration by regulating Schwann cell activation and axon elongation

Schwann cells in peripheral nerves react to traumatic nerve injury by attempting to grow and regenerate.Howeve r,it is unclear what factors play a role in this process.In this study,we searched a GEO database and found that expression of platelet factor 4 was markedly up-regulated after sciatic nerve injury.Platelet factor is an important molecule in cell apoptosis,diffe rentiation,survival,and proliferation.Further,polymerase chain reaction and immunohistochemical staining confirmed the change in platelet factor 4 in the sciatic nerve at different time points after injury.Enzyme-linked immunosorbent assay confirmed that platelet factor 4 was secreted by Schwann cells.We also found that silencing platelet factor 4 decreased the proliferation and migration of primary cultured Schwann cells,while exogenously applied platelet factor 4 stimulated Schwann cell prolife ration and migration and neuronal axon growth.Furthermore,knocking out platelet factor 4 inhibited the prolife ration of Schwann cells in injured rat sciatic nerve.These findings suggest that Schwann cell-secreted platelet factor 4 may facilitate peripheral nerve repair and regeneration by regulating Schwann cell activation and axon growth.Thus,platelet factor 4 may be a potential therapeutic target for traumatic peripheral nerve injury.

外周血CD4^(+)PD-1^(+)Tcells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性分析

目的 探讨外周血CD4^(+)程序性细胞死亡受体-1(PD-1)^(+)T cells及CD4^(+)T淋巴细胞三磷酸腺苷(ATP)含量与复发性卵巢癌疗效的相关性。方法 选取30例复发性卵巢癌患者为复发组,30例未复发卵巢癌患者为非复发组;另选取30名同期体检健康者作为对照组。评估外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效的相关性。结果 复发组和非复发组的CD4^(+)PD-1^(+)T cells较对照组明显升高(P<0.05)。复发组和非复发组的CD4^(+)T淋巴细胞ATP含量较对照组明显降低(P<0.05)。复发组治疗后CD4^(+)PD-1^(+)Tcells显著低于治疗前(P<0.05),治疗后CD4^(+)T淋巴细胞ATP含量显著高于治疗前(P<0.05)。CD4^(+)PD-1^(+)T cells与复发性卵巢癌疗效成负相关(r=-0.393,P=0.039),CD4^(+)T淋巴细胞ATP含量与复发性卵巢癌疗效成正相关(r=0.449,P=0.031)。结论 复发性卵巢癌患者外周血CD4^(+)PD-1^(+)T cells及CD4^(+)T淋巴细胞ATP含量与疗效密切相关。

Targeted migration of mesenchymal stem cells modified with CXCR4 to acute failing liver improves liver regeneration

AIM: To improve the colonization rate of transplanted mesenchymal stem cells (MSCs) in the liver and effect of MSC transplantation for acute liver failure (ALF).

Expression and Immune Effect of Toll-Like Receptor 4 in Human Trophoblast Cells

This study investigated the expression and immune effect of TLR4 in human trophoblast cells. The expression level of TLR4 mRNA in normal and LPS-stimulated human term trophoblast cells （1 mg/L LPS, 12 h） was detected by RT-PCR. In LPS-stimulated human term trophoblast cells of TLR4-blocked group and non-TLR4-blocked group, and normal term trophoblast cells of blank control group, apoptosis rate was measured by flow cytometry （FCM）, and the level of TNF-α determined by using enzyme linked immunosorbent assay （ELISA） respectively. RT-PCR results showed that the expression level of TLR4 mRNA in LPS-stimulated human trophoblast cells was significantly higher than that in normal cells （P〈0.01）. FCM revealed that there was significant difference in apoptosis rate of LPS-stimulated human term trophoblast cells between TLR4-blocked group and non-TLR4-blocked group （P〈0.05）, or between TLR4 antibody-blocked group and blank control group. ELISA indicated that the level of TNF-α in LPS-stimulated human trophoblast cells also had statistical differences between TLR4 antibody-blocked group and non-TLR4 antibody-blocked group （P〈0.05）. Our results suggest that TLR4 plays an important role in the immunological mechanism of apoptosis and secretion of TNF-α of human term trophoblast cells stimulated by LPS.

HNF-4α determines hepatic differentiation of human mesenchymal stem cells from bone marrow

AIM: To investigate the differentiation status and key factors to facilitate hepatic differentiation of human bone-marrow-derived mesenchymal stem cells (MSCs). METHODS: Human MSCs derived from bone marrow were induced into hepatocyte-like cells following a previously published protocol. The differentiation status of the hepatocyte-like cells was compared with various human hepatoma cell lines. Overexpression of hepatocyte nuclear factor (HNF)-4α was mediated by adenovirus infection of these hepatocyte-like cells. The expression of interesting genes was then examined by either re-verse transcription-polymerase chain reaction (RT-PCR) or real-time RT-PCR methods. RESULTS: Our results demonstrated that the differentiation status of hepatocyte-like cells induced from human MSCs was relatively similar to poorly differentiated human hepatoma cell lines. Interestingly, the HNF-4 isoform in induced MSCs and poorly differentiated human hepatoma cell lines was identified as HNF4γ instead of HNF-4α. Overexpression of HNF-4α in induced MSCs significantly enhanced the expression level of hepatic-specific genes, liver-enriched transcription factors, and cytochrome P450 (P450) genes. CONCLUSION: Overexpression of HNF-4α improves the hepatic differentiation of human MSCs from bone marrow and is a simple way of providing better cell sources for clinical applications.

Amygdalin inhibits genes related to cell cycle in SNU-C4 human colon cancer cells

AIM： The genes were divided into seven categories according to biological function; apoptosis-related, immune response-related, signal transduction-related, cell cyclerelated, cell growth-related, stress response-related and transcription-related genes. METHODS： We compared the gene expression profiles of SNU-C4 cells between amygdalin-treated （5 mg/mL, 24 h） and non-treated groups using cDNA microarray analysis. We selected genes downregulated in cDNA microarray and investigated mRNA levels of the genes by RT- PC R. RESULTS： Microarray showed that amygdalin downregulated especially genes belonging to cell cycle category： exonuclease 1 （EXO1）, ATP-binding cassette, sub-family F, member 2 （ABCF2）, MRE11 meiotic recombination 11 homolog A （MRE11A）, topoisomerase （DNA） Ⅰ （TOP1）, and FK506 binding protein 12-rapamycin-associated protein 1 （FRAP1）. RT-PCR analysis revealed that mRNA levels of these genes were also decreased by amygdalin treatment in SNU-C4 human colon cancer cells. CONCLUSION： These results suggest that amygdalin have an anticancer effect via downregulation of cell cycle-related genes in SNU-C4 human colon cancer cells, and might be used for therapeutic anticancer drug.

Transfection with CXCR4 potentiates homing of mesenchymal stem cells in vitro and therapy of diabetic retinopathy in vivo

AIM：To investigate the effect of the overexpression of C-X-C chemokine receptor type 4（CXCR4） on homing of mesenchymal stem cells（MSCs） in vitro and therapeutic effects of diabetic retinopathy（DR） in vivo.METHODS：MSCs were infected by lentivirus constructed with CXCR4.The expression of CXCR4 was examined by immunofluorescence,Western blot,and quantitative polymerase chain reaction.CXCR4-overexpressing MSCs were cultured in vitro to evaluate their chemotaxis,migration,and apoptotic activities.CXCR4-overexpressing MSCs were intravitreally injected to observe and compare their effects in a mouse model of DR.The histological structure of DR in rats was inspected by hematoxylin and eosin staining.The expression of rhodopsin,neuron-specific enolase（NSE）,and inflammatory cytokines interleukin（IL）-6 and tumor necrosis factor（TNF）-α was examined by Western blot and immunohistochemical analyses.RESULTS：The transduction of MSCs by lentivirus was effective,and the transduced MSCs had high expression levels of CXCR4 gene and protein.Improved migration activities were observed in CXCR4-overexpressing MSCs.Further,reduced retinal damage,upregulation of rhodopsin and NSE protein,and downregulation of inflammatory cytokines IL-6 and TNF-α were observed in CXCR4-overexpressing MSCs in vivo.CONCLUSION：The homing of MSCs can be enhanced by upregulating CXCR4 levels,possibly improving histological structures of DR.CXCR4-overexpressing MSCs can be a novel strategy for treating DR.

Enhancement of insulin-producing cell differentiation from embryonic stem cells using pax4-nucleofection method

AIM： To enhance the differentiation of insulin producing cell （IPC） ability from embryonic stem （ES） cells in vitro. METHODS： Four-day embryoid body （EB）-formatted ES cells were dissociated as single cells for the followed plasmid DNA delivery. The use of NucleofectorTM electroporator （Amaxa biosystems, Germany） in combination with medium-contained G418 provided a high efficiency of gene delivery for advanced selection. Neucleofected cells were plated on the top of fibronectincoated Petri dishes. Addition of Ly294002 and raised the glucose in medium at 24 h before examination.The differentiation status of these cells was monitored by semi-quantitative PCR （SQ-PCR） detection of the expression of relative genes, such as oct-4, sox-17, foxa2, mixll, pdx-1, insulin 1, glucagons and somatostatin. The percentage of IPC population on d 18 of the experiment was investigated by immunohistochemistry （IHC）, and the content/secretion of insulin was estimated by ELISA assay. The mice with severe combined immunodeficiency disease （SCID） pretreated with streptozotocin （STZ） were used to eliminate plasma glucose restoration after pax4^＋ ES implantation. RESULTS： A high efficiency of gene delivery was demonstrated when neucleofection was used in the present study; approximately 70% cells showed DsRed expression 2 d after neucleofection. By selection of medium-contained G418, the percentage of DsRed expressing cells kept high till the end of study. The pancreatic differentiation seemed to be accelerated by pax4 nucleofection. When compared to the group of cells with mock control, foxa2, mixll, pdxl, higher insulin and somatostatin levels were detected by SQ-PCR 4 d after nucleofection in the group of pax4 expressing plasmid delivery. Approximately 55% of neucleofected cells showed insulin expression 18 d after neucleofection, and only 18% of cells showed insulin expression in mock control. The disturbance was shown by nucleofected pax4 RNAi vector; only 8% of cells expressed insulin 18 d after nucleofection. A higher IPC population was also detected in the insulin content by ELISA assay, and the glucose dependency was demonstrated in insulin secretion level. In the animal model, improvement of average plasma glucose concentration was observed in the group of pax-4 expressed ES of SCID mice pretreated with STZ, but no significant difference was observed in the group of STZ-pretreated SCID mice who were transplanted ES with mock plasmid. CONCLUSION： Enhancement of IPC differentiation from EB-dissociated ES cells can be revealed by simply using pax4 expressing plasrnid delivery. Not only more IPCs but also pancreatic differentiation-related genes can be detected by SQ-PCR. Expression of relative genes, such as foxa 2, mixl 1, pdx-1, insulin 1 and somatostatin after nucleofection, suggests that pax4 accelerates the whole differentiation progress. The higher insulin production with glucose dependent modulation suggests that pax4 expression can drive more mature IPCs. Although further determination of the entire mechanism is required, the potential of pax-4-nucleofected cells in medical treatment is promising.