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Amyotrophic lateral sclerosis is a neurodegenerative disease caused by the loss of motor neurons in the brain and spinal cord. There is currently no effective cure. The emergence of gene therapy brings hope to treatment, which can be achieved by delivering transgenes to replace or correct defective genes, as well as the expression of neurotrophic factors. The vectors of gene therapy can be viral vectors and non-viral vectors. Lentiviral vectors can be used to deliver therapeutic sequences to motor neurons in the central nervous system. Adeno-associated viruses can effectively mediate gene expression and delivery of neurotrophic factors. Gene editing and antisense oligonucleotides therapy are also perspective treatment options. This article summarizes gene therapy for amyotrophic lateral sclerosis from basic experiments and clinical trials.
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Gene therapy is expected to restore the function of genetic material fundamentally and it has become a new trend in inherited retinal dystrophy treatment.Antisense oligonucleotide (AON) is a kind of small molecule nucleic acid drug, which can specifically bind to messenger RNA through the base pairing principle, thus interfering or modifying gene expression at the transcription and translation level.Possessing the advantages of high specificity and efficiency, wide targeting range, low immunogenicity and limited adverse effect, AON has become a novel remedy for inherited retinal dystrophy.Currently, three different AON drugs have already been used in clinical trials for inherited retinal dystrophy.In this review, the chemical structure modification, properties and mechanism of AON, and the therapeutic strategies of AON in different inherited retinal dystrophy diseases in recent years were summarized.
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Objective To investigate the effect of antisense oligonucleotides of miRNA-34a on non-small cell lung cancer (NSCLC) and its molecular mechanism.Methods The expression of miRNA34a in human non-small cell lung cancer cell line HCC827 and human normal lung cell MRC-5 was detected by real time fluorescence quantitative polymerase chain reaction (qRT-PCR).HCC827 cells were divided into three groups:blank control group,negative control group,anti-sense oligonucleotide group (liposome 2000 transfected anti-sense oligonucleotide miRNA-34a);cell counting kit-8 (CCK-8) method was used to detect cell proliferation,Jimsa staining was used to detect cell cloning ability,Transwell test was used to detect cell migration and invasion ability;RT-PCR and Western blot were used to detect phosphatase and tensin homolog (PTEN),phosphorylation-protein kinase B (p-Akt),phosphatidylinositol-3-kinase (PI3K)mRNA and protein expression.Results The relative expression of miRNA34a in HCC827 cells was significantly higher than that in human normal lung cells (P ＜ 0.01).The relative expression of miRNA34a in antisense oligonucleotide miRNA-34a group was significantly lower than that of negative control group and blank control group (P ＜ 0.05),and there was no significant difference between negative control group and blank control group (P ＞ 0.05).At 48 h,72 h and 96 h,the proliferation level of HCC827 cells in antisense oligonucleotide miRNA-34a group was significantly lower than that in negative control group and blank control group (P ＜ 0.05).The cell cloning rate of antisense oligonucleotide miRNA-34a group was significantly lower than that of negative control group and blank control group (P ＜ 0.01).The number of migration and invasion of HCC827 cells in antisense oligonucleotide RNA-34a group was significantly lower than that in negative control group and blank control group (P ＜0.01).The relative expression of PTEN mRNA and protein in antisense oligonucleotide miRNA-34a group was significantly higher than that in negative control group and blank control group (P ＜ 0.05);the relative expression of p-Akt,PI3K mRNA and protein in antisense oligonucleotide miRNA-34a group were significantly lower than that in negative control group and blank control group (P ＜ 0.05).Conclusions The expression level of miRNA-34a in human nonsmall cell lung cancer cells is significantly higher than that in human normal lung cells.Antisense oligonucleotides of miRNA-34a can inhibit the proliferation,cloning,migration and invasion of human non-small cell lung cancer cells.The mechanism may be related to the negative regulation of PTEN/p-Akt/PI3K signaling pathway.
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Objective To investigate the effect of microRNA-21 (miR-21) antisense oligonucleoti-de on collagen synthesis in the rat hepatic satellite cells ( HSCs) .Methods Rat hepatic stellate cells were isolated and cultured; the miR-21 antisense oligonucleotide was transfected into HSCs with lipofectamine 2000;after incubation 48 h, the HSCs were collected.The expression of miR-21 was detected with reverse transcription polymerase chain reaction ( RT-PCR) , andα-smooth muscle actin (α-SMA) protein in HSCs with Western blot.The cell proliferation was assayed with methyl thiazolyl tetrazolium ( MTT) method.Re-sults Compared to scrambled control group, the expression of miR-21 was reduced by 76%( P <0.01), the proliferation activity of HSCs was reduced by(26 ±3)%( P <0.01), the expressions of type I and III collagen proteins were reduced by(61 ±7)%and (48 ±6)%( P <0.01).Conclusions The miR-21 an-tisense oligonucleotide could reduce significantly the expression of miR-21, and inhibit HSC proliferation and extracellular matrix production.
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BACKGROUND:Currently, antisense oligonucleotides (AS-ODN) have a good prospect in gene therapy, but AS-ODN with smal molecular weight cannot easily enter into the cels, which is susceptible to nuclease degradation. Therefore, there is stil a lack of fundamental understanding about how to improve their transfection efficiency, and target-based transferring. OBJECTIVE:To investigate whether a weak cationic and phosphorylcholine-containing diblock copolymer (MPC30-DEA70) can act as a carrier system to deliver a chemicaly synthesized transforming growth factor-β1 (TGF-β1) AS-ODN into myocardial cels. METHODS: MPC30-DEA70 was compounded with TGF-β1 AS-ODN at various N/P ratios and the MPC30-DEA70/TGF-β1 AS-ODN complexes were characterized by DNA electrophoresis. MTT assay was used to observe the biocompatibility. Confocal laser scanning microscope was used to observe the distribution and location of MPC30- DEA70/TGF-β1 AS-ODN in cells. Flow cytometry was used to detect the transfection efficiency and fluorescence intensity of MPC30-DEA70/TGF-β1 AS-ODN in cells. Western blot and RT-PCR methods were employed to measure the expression of TGF-β1 in cells. RESULTS AND CONCLUSION: Cell growth inhibition showed that the MPC30-DEA70 had low cytotoxicity to myocardial cells within the effective transfection dosage range (< 20 mg/L). Data from the flow cytometry test indicated a clear trend of increasing transfection efficiency with the increasing of N/P ratios. At high N/P ratios, the expression levels of TGF-β1 mRNA and protein in myocardial cells were significantly lower. This study shows that MPC30-DEA70 can work as an effective transgenic vector in myocardial cells. TGF-β1 AS-ODN can silence the expression of TGF-β1 gene efficiently and specially, and may antagonize TGF-β1-mediated biological function.
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Objective To investigate the effect of mouse double minute 2 (MDM2) mRNA ASON and mismatched oligonucleotide (ASONM) radiolabeled with 99Tcm on target gene expression in LNCaP cells.Methods The ASON and ASONM targeted to MDM2 mRNA were synthesized and radiolabeled by 99Tcm with the bifunctional chelator of HYNIC.The labeling efficiency,radiochemical purity,stability and molecular hybridization activity were investigated.The different concentrations of 99Tcm-HYNIC-ASON (0,100,500 nmol/L) and 99Tcm-HYNIC-ASONM (500 nmol/L) coated with lipofectamin 2000 were incubated with prostate cancer cells for 24 h,then RT-PCR and Western blot were carried out to assay the MDM2,p53 mRNA and the corresponding protein level.The variables of RT-PCR and Western blot were analyzed using one-way analysis of variance and q test.Results The labeling efficiency of ASON and ASONM were (65.15± 2.05)％ (n=5) and (64.93±2.18)％ (n=5),respectively.The radiochemical purity were both more than 90％.99Tcm-HYNIC-ASON had a good stability and could hybridize to the sense oligonucleotide (SON).The contents of MDM2 mRNA in 0,100,500 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups were 0.458±0.035,0.250±0.026,0.174±0.032,0.463±0.033,respectively,and there were significant differences between each 2 groups except between 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM groups (F=33.69,q =24.32-91.45,all P＜0.01).The average density of MDM2 protein in the 4 groups were 90.712±3.042,71.218±2.915,32.775±3.062,88.121±2.710,respectively (F=235.93,q=6.43-19.14,all P＜0.01; except 0 nmol/L99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).The contents of p53 mRNA in the 4 groups were 0.185±0.046,0.203±0.040,0.213±0.027,0.163±0.049,respectively(F =2.18,P＞ 0.05).The average density of p53 protein was 33.865 ± 2.213,70.445±2.180,99.025±3.012,38.351±3.271,respectively (F=53.98,q =3.32-6.74,all P＜0.01 ; except 0 nmol/L 99Tcm-HYNIC-ASON and 500 nmol/L 99Tcm-HYNIC-ASONM).Conclusions The MDM2 antisense probe can accumulate in the prostate cancer cells,and specially hybridize to the MDM2 mRNA and inhibit target gene expression.This novel molecular probe has a promising potential for the diagnosis of prostate cancer at gene level.
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Objeetive To explore the value of antisense imaging of 99Tcm-labeled ASON targeting mouse double minute 2(MDM2) mRNA for the diagnosis of human prostate cancer.Methods The ASON targeting MDM2 mRNA and the mismatched oligonucleotide (ASONM) were synthesized and radiolabeled with 99Tcm using the bifunctional chelator HYNIC.The labeling efficiency and radiochemical purity were investigated.Animal models of nude mice bearing human prostate cancer LNCaP were established and divided into 3 groups with 10 mice in each group.99Tcm-HYNIC-ASON,99Tcm-HYNIC-ASONM (study groups) and 99TcmO4-(control group) were injected at the dose of 7.4 MBq through the tail vein,respectively.Tumor imaging was acquired with SPECT and the tumor-to-muscle (T/M) ratio was measured.The data was compared by one-way analysis of variance.Results The labeling efficiencies of ASON and ASONM were (65.15± 2.05) ％ and (64.93±2.18) ％,respectively.Their radiochemical purity was greater than 90％.At 1,4 and 10 h post injection,the T/M ratios of 99Tcm-HYNIC-ASON group were 3.217±0.125,3.749± 0.201 and 4.028±0.186,and those of 99Tcm-HYNIC-ASONM group were 1.579t0.128,1.715±1.140 and 1.683±0.139,and control group 2.146±0.132,1.847±0.124,1.528±0.152,respectively.The T/M ratios in control group and 99Tcm-HYNIC-ASONM group were significantly lower than those in 99Tcm-HYNICASON group at 1,4 and 10 h,respectively (F=213.37-235.41,t=3.527-4.738; all P＜0.01).The T/M ratios of 99Tcm-HYNIC-ASONM group and control group were not significantly different at 1,4 and 10 h (t=2.154,2.287 and 2.236,all P＞0.05).Conclusion The antisense probe of MDM2 can accumulate specifically in prostate cancer tissue in animal models,which might be useful as a non-invasive genetic tool for the early diagnosis of prostate cancer.
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Objective To investigate effect of FK506 binding protein 51 (FKBP51) on the c-JunN-terminal kinase (JNK) pathway in cerebral ischemia-reperfusion injury.Methods Transient global cerebral ischemia rat models were made by four-vessel method.Healthy male SD (Sprague Dawley) rats were randomly divided into:sham group,ischemia/reperfusion group (I/R group),FKBP51 antisense oligonucleotide group (FKBP51 ASODN group),FKBP51 missense oligonucleotide group (FKBP51 MSODN group),and solvent control group (TE group).The effect of FKBP51 ASODN on expression of FKBP51 protein and JNK was detected,and c-Jun phosphorylation was detected by Western blot.Results (1) FKBP51 protein expression in the FKBP51 ASODN group was reduced.The change of FKBP51 protein expression between the FKBP51 ASODN and sham groups was statistically significant (P ＜ 0.05).(2) The expression differences of total JNK protein between all the groups were not statistically significant (P ＞ 0.05).The expression of p-JNK in sham group was significantly less than the other groups (P ＜ 0.05).The expressions of p-JNK in I/R 3d,TE,and FKBP51 MSODN groups were higher relative to Sham group; however,the differences among those three groups were not statistically significant (P ＞ 0.05).The expression of p-JNK in FKBP51 ASODN group was significantly less than FKBP51 MSODN group (P ＜ 0.05).(3) The expression differences of total c-Jun protein among all groups were not statistically significant (P ＞ 0.05).The expression of p-c-Jun in sham group was significantly less than the other groups (P ＜ O.05).The expressions of p-c-Jun in I/R 6 h,TE,and FKBP51 MSODN groups were higher relative to the sham group; however,the differences among those three groups were not statistically significant (P ＞ 0.05).The expressions of p-c-Jun in FKBP51 ASODN group was significantly less than FKBP51 MSODN group (P ＜ 0.05).Conclusions FKBP51 might activate JNK signaling pathway in cerebral ischemia-reperfusion injury.
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[ABSTRACT]AIM:ToinvestigatetheroleoftinyantisensenucleicacidagainstmiR-155(tinyantimiR-155, t-antimiR-155) in multiple myeloma cells .METHODS:According to the seed sequence of miR-155, t-antimiR-155 was designed and synthesized .t-antimiR-155 was transfected by Lipofectamine TM 2000 into RPMI-8266 cells.The cells were di-vided into t-antimiR-155 group, scrambled control (SCR) group and blank control group .The growth-inhibitory potencies were measured by MTT assay .The ability of cell colony formation was detected by cell colony formation assay .The cell ap-optosis was assessed by flow cytometry with annexin V /PI double staining .RESULTS: The best concentration and time were 0.4 μmol/L and 48 h, respectively.The cell colony forming experiment showed that the circumstances of forming cell community in t-antimiR-155 group was weaker than that in SCR group , and the colony formation inhibitory rate of former was significant higher than the latter .Compared with SCR group , the cell apoptosis in t-antimiR-155 group significantly in-creased.CONCLUSION: The t-antimiR-155 inhibits the progression of multiple myeloma cells by interfering with miR-155.miR-155 may serve as a potential target in gene therapy for treating multiple myeloma .
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Objective To study the inhibitory effect of knocking down miR-30a-5p on the U87 human glioma xenograft growth and its possible mechanism.Methods Nude mice bearing subcutaneous U87 human glioblastoma were established and separated into three groups (eight for each group) by randomized digital table method,including control group,scr-ODN treated group and AS-miR-30a-5p treated group.After relevant subcutaneous injection treatment,tumor size was measured every other day until the observation period ended.Researchers executed the animals after the treatment,stripped tumor tissues and extracted RNA and protein.Real-time PCR was conducted to detect the expression of miR-30a-5p.The histopathological characteristics and proliferation and apoptosis biological characters (including SEPT7,PCNA,cyclin D1,MMP-2,apoptosis related factor P53,bcl-2 and caspase3) were evaluated by HE and immunohistochemical staining,Westem blot analysis respectively,and the cell apoptosis was detected by TUNEL method.Results In AS-miR-30a-5p treated group,the tumor growth was delayed and the final tumor volume was smaller than that in the control and scr-ODN treated group (F =7.167,P ＜0.05),and the expression of miR-30a-5p was knocked down.The expression of PCNA,cyclin D1 were significantly downregulated while P53,SEPT7 and caspase3 up-regulated.Apoptotic index was increased significantly.Conclusion As-miR-30a-5p suppresses the growth of U87 human gliomas xenografts significantly.Malignant phenotype of tumors are reversed to a considerable degree.Therefore,miR-30a-5p can be a candidate for targeted therapy of human glioma.
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ObjectiveTo confirm the effect of miR-93 inhibitor in glioma cell growth and invasion.MethodsMalignant glioma cells were transfected with miR-93 inhibitor by lipofectamin to downregulate their overexpression of miR-93.Real time-PCR was taken to measure miR-93 expression after transfection.The cell cycle kinetics and cell growth rate were detected by flowcytometry and MTT assay,the cell proliferative ability was evaluated by soft agar assay,and the invasive ability was detected by transwell assay.ResultsThe highlevel expression of miR-93 was downregulated effectively in glioma cells after transfecting the miR-93 inhibitor.Meanwhile,the cell cycle progress was delayed,S phase cells were reduced,the speed of growth was slowed,cloning formation ability was receded,the number of cells through the matrigel was reduced,and invasive ability was significantly repressed.ConclusionDownregulation of miR-93 expression could inhibit the proliferative ability and invasive ability of glioma cells.
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Objective To create Mucin gene 1 (MUC1) antisense peptide nucleic acid (PNA),and to observe its effects on MKN-45 cell invasion and explore the mechanism. Methods The sequence of antisense PNA was designed according to MUC1 gene sequence and transfected into human gastric cancer cells (MKN-45) by liposome,and the empty vector group (randomized control group)and blank control group (negative control group) were involved. The expression of MUC1 was detected by real time quantitative PCR and the changes of E-cadherin expression were also observed.The effects on gastric cancer cell invasion were tested with transwell chamber assays.Results The expression of MUC1 gene was effectively suppressed by the 3 created antisense PNA,and their expression level (0.62±0.18,0.49±0.12 and 0.60±0.21) was significantly lower than that of negative control group (1.18 ± 0.03,P ＜ 0.01). There was no significant difference between radomized control group and negative control group (1.00±0.04,P=0.657).After MUC1 PNA transfected,the capability of gastric cancer cell invasion decreased significantly (P=0.005).And the expression of E-cadherin at mRNA and protein level was up-regulated.Conclusions There is negative correlation between MUC1 and E-cadherin expression in gastric cancer cell MKN-45.The capability of tumor cell invasion is significantly inhibited by suppressing MUC1 gene expression.
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Objective To investigate the effects of repeated intrathecal cyclic AMP response elementbinding protein (CREB) antisense oligodeoxynucleotide (ODN) on the expression of NR2A in spinal cord in mice with neuropathic pain produced by chronic constrictive injury of the sciatic nerve (CCI).Methods Forty C57BL/6 male mice in which intrathecal catheter was successfully implanted were randomly divided into 4 groups ( n =10 each):normal saline group (group NS),CREB sense ODN group (group S),CREB missense ODN group (group M),and CREB antisense ODN group (group A).In groups NS,S,M and A,normal saline 5μl,sense ODN 5 μg/5 μl,missense ODN 5 μg/5 μl and antisense ODN 5 μg/5 μl were injected intrathecally once a day for 6 days,starting from the 1st day after CCI,respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured on day 1 before CCI and on day 1,3,5 and 7 after CCI.Five mice from each group were sacrificed on day 7 and 14 after CCI and the lumbar segment of the spinal cord (L3-5 )was removed for determination of NR2A expression using Western blot and RT-PCR.Results Compared with the baseline value,no significant change was found in PWMT and PWTL on day 1-7 after CCI in group A ( P ＞0.05),while PWMT and PWTL were significantly decreased on day 1-7 after CCI in groups NS,S and M (P ＜0.05).Compared with groups NS,S and M,the expression of NR2A mRNA and protein was significantly downregulated on day 7 and 14 after CCI in group A ( P ＜ 0.05).The expression of NR2A mRNA and protein was significantly up-regulated on day 14 after CCI compared with that on day 7 after CCI in all the groups.Conclusion Intrathecal CREB antisense ODN during the development of neuropathic pain can attenuate neuropathic pain and inhibition of the expression of NR2A in mouse spinal cord may be involved in the mechanism.
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As estatinas são produtos farmacêuticos de grande sucesso em todo mundo. Entretanto, muitos pacientes, ou por não as tolerarem adequadamente, ou por necessitarem de taxas mais baixas de LDL-colesterol estabelecidas como metas, poderão ter benefícios clínicos com o emprego de novos medicamentos. Numerosas linhas de pesquisa encontram-se em evolução, avaliando produtos com atuação em diferentes vias moleculares: inibidores de síntese da apolipoproteína B, inibidores da DGAT2, da ACAT2, da MTP, da esqualeno sintase, tireoidemiméticos e inibidores da PCSK9. Espera-se, para futuro próximo, a introdução no mercado desses medicamentos, que poderão auxiliar ainda mais na prevenção primária e secundária da doença aterosclerótica coronária, flagelo deste novo século.
Statins are pharmaceutical products that obtained worldwide success. However, some patients with inadequate tolerability to these medications and the need of achieving lower LDL-cholesterol levels as recommended targets, may receive clinical benefits with the use of new drugs. Many research lines have been in evolution evaluating products that act in different molecular pathways: inhibitors of apoliprotein B synthesis, inhibitors of DGAT2, ACAT2, MTP, squalene synthase, thyromimetics, and inhibitors of PCSK9. It is a hope that the future introduction of many of these products on the market will help furthermore primary and secondary prevention of coronary heart disease, a scourge of this new century.
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Humans , Apolipoproteins B/analysis , Atherosclerosis/complications , Atherosclerosis/mortality , Cholesterol, LDL/analysis , Cardiovascular Diseases/complications , Cardiovascular Diseases/mortality , Thyroid Hormones/analysis , Oligonucleotides, Antisense , Proprotein ConvertasesABSTRACT
Objective To investigate the role of microRNA-21 (miRNA-21) in promoting proliferation of Kazakh's esophageal squamous cell carcinoma (ESCC) cell line Eca109 and Kazakh's ESCC tissues, and its effects on the expression of programmed cell death 4(PDCD4)gene.Methods TheEca109cellsweredividedintomiRNA-21Mimicsgroup(transfersequence:5′-UCAACAUCAGUCUGAUAAGCUA-3′),miRNA-21inhibitorgroup(transfersequence:5′-UAGCUUAUCAGACUGAUGUUGA-3′), negative control group (the transfer random sequence)and normal control group (no transfection).The cells were seeded at 4.5 × 105/well in 6-well cell culture plates. According to RNA interference technology,thecells were transfected with LipofectamineTM 2000. After transfection, the cell proliferation was determined by cell number counting.The expression of miRNA-21 was detected by real-time quantitative polymerase chain reaction (qRT-PCR), and the expression of PDCD4 protein was measured by Western blot.Meanwhile, the expression of miRNA-21 and PDCD4 protein were performed in 18 pairs of Kazakh's ESCC and corresponding adjacent normal esophageal mucosa.ResultsAt 48 hour after transfection,compared with normal control group, the cell number increased 36％ (P=0.002) in miRNA-21 Mimics group, decreased 28％ (P=0.002) in miRNA-21 inhibitor group, and did not change significantly in negative control group (P=0.515). At 48 hour after transfection, the relative expression of miRNA-21 in miRNA-21 Mimics group, miRNA-21 inhibitor group, negative control group and normal controlgroup was 0.37±0.10, 9.17± 1.08, 0.74±0.23 and 1.04±0.34,respectively, and the relative protein expression of PDCD4 was 1.47 ± 0.11, 0.61±0.09, 0.89 ±0.12 and 0.79±0.02 accordingly.Compared with normal control group, the relative expression of miRNA-21 decreased (P=0.031) and the relative protein expression of PDCD4 up regulated (P=0.001) in miRNA-21 inhibitor group, the relative expression of miRNA-21 increased (P=0.001) and the relative protein expression of PDCD4 down regulated (P=0.030) in miRNA-21 Mimics group,and there was no significant difference in negative control group (P=0.272 and 0.541).In 16 pairs of Kazakh's ESCC tissues, the expression of miRNA-21 was significantly higher in tumor tissues (0.11 ±0.09) than that of corresponding adjacent esophageal normal tissues (0.03 ± 0.03, P=0.001), while the relative protein expression of PDCD4 was significantly lower (0.92 ± 0.39) than that of corresponding adjacent normal esophageal tissues (1.57 ± 0.80, P=0.004). And the higher expression of miRNA-21, the lower relative protein expression of PDCD4 (r=-0.538, P=0.046).ConclusionmiRNA-21 may promoted the cell proliferation through inhibiting PDCD4 expression,which involved in the pathogenesis of Kazakh's ESCC.
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Objective To investigate the effect of VEGF ASODN in vitro and in vivo on the biological characteristics of human prostate cancer PC3 cells and its effect in xenotransplanted tumors in nude mice by local ASODN injection.MethodsVEGF ASODN was delivered into PC3 cells by Oligofectamine.There were three experimental groups: VEGF ASODN,VEGF ODN and control.Soft agar assay and matrigel invasion assay were used to measure cellular transformation and invasion ability,respectively.Tumor formation assay in nude mice was used to evaluate the effect of VEGF ASODN on proliferation of PC3 cells in vivo.The xenotransplanted prostate tumor model in nude mice was established and the effect of local ASODN injection on the inhibition of tumor growth in vivo was examed.ResultsThe soft agar colony numbers for control,ODN,and ASODN treated cells were 53.67±5.86,52.33±6.43 and 26.00±4.58,respectively (F =13.73,P＜0.01).The numbers of invaded cells for three group were 45.60±5.53,42.35±6.21 and 18.37±3.52,respectively (F =14.18,P ＜0.01).Tumor cells transfected with VEGF ASODN proliferated more slowly than other groups.28 days later after tumor cells were injected into nude mice,the tumor sizes of three groups were (1330.32±81.38) mm3,(1267.64±120.26) mm3 and (641.83±58.34) mm3 (F =17.26,P ＜0.01).After treating the transplanted tumor with VEGF ASODN or control oligos for four weeks,the tumor weight of three groups was (1.25±0.08) g,(1.17±0.06) g and (0.41±0.05) g,respectively.Comparing with control groups,the tumor inhibitory rates of ODN group and ASODN group were 6.4 ％ and 67.2 ％,respectively (x2=17.72,P＜0.005).Conclusion VEGF ASODN could inhibit VEGF expression in PC3 cells and lead to increasing cell apoptosis.After VEGF ASODN treatment,tumorigenesis in vitro is inhibited and cell invasion ability is decreased.The tumors originated from cells transfected with VEGF ASODN grow more slowly than control groups.Also local injection of VEGF ASODN could inhibit the growth of transplanted tumors in nude mice.
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Objective To investigate the effects of Livin antisense oligonucleotide (ASODN) on the proliferation and apoptosis of human leukemia (K562) cells. Methods Specific phosphorothioate ASODN and missense oligonucleotide (MSODN) target Livin mRNA were synthesized and transfected into K562 cells following cationic liposome. The proliferation inhibition of K562 cells was assessed by MTT. The apoptosis rate of each group was detected by Annexin V-FITC. The expression of Livin mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR). Results ASODN at a final concentration of 600 nmol/Lcould inhibit the K562 cells proliferation (IR) was (52.99t2.67) % and the expressions of Livin mRNA (ODR)was (59.75±3.24) %, the apoptosis rate was apparently increased [(36.89±1.08) %] (P ＜0.01); but the difference between Lip-MSODN group, Lip control group and cell control group was not statistically significant (P ＞0.05).Conclusion Livin ASODN may decrease Livin gene expression, suppress K562 cells proliferation effectively, and induce significant apoptosis of K562 cells.
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Objective: To explore the effects of antisense oligonucleotide (ASODN) of connective tissue growth factor (CTGF) on the apoptosis of human hypertrophic scar fibroblasts (HSF) and the related mechanisms. Methods: The hypertrophic scar fibroblasts were cultured in vitro and were divided into the following 3 groups: CTGF-ASODN-treated group, liposome control group and blank control group. The distribution of CTGF-ASODN in HSF of different phases was observed under fluorescent microscope in 3 groups. Flow cytometer was employed to detect the apoptotic rate of HSF and the expression of Fas. The expression of CTGF, Fas and bcl-2 mRNA in HSF was assessed by RT-PCR. Results: 24 h after transfection, large quantity of yellow-green fluorescence was seen in the HSF cytoplasm of CTGF-ASDON group in a scattered or doted manner, but not found in that of the other 2 groups. The apoptosis rate of HSF in CTGF-ASDON group was significantly higher than those in the other 2 groups (P<0.01). RT-PCR result showed that CTGF mRNA expression in transfection group was significantly lower than that in the blank control group(P<0.05) and the Fas mRNA expression was significantly higher than that in the blank control group(P<0.01); the expression of bcl-2 mRNA had no significant difference between the 2 groups. Conclusion: CTGF-ASODN can promote the apoptosis of HSF in vitro, which might be related to the up-regulation Fas gene.
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Objective To investigate the effect of antisense oligonucleofide(PCNA ASON)and vascuiar endothelial growth factor (VEGF) gene traneduction on restenosis after balloon angiopasty.Memods chinese rabbits were randomly divided into control group(I),PCNA ASON(II),VEGF only (Ⅲ),PCNA ASON+VEGF(Ⅳ)groups.Each group included 7 rabbits.Restenosis wasevaluated by pathology immunohistochemistry and Western-blotting for the expression of PCNA,and the depth and area oftunica media and tunicca intima were measured. Results All rabbits experienced restenosis on different severities,especially in control group.Lesions were less severe in PCNA ASON and VEGF groups than in controls.The proliferation of smooth muscle and intima significantly ameliorated in PCNA ASON and VEGF combination group that in PCNA ASON or VEGF groups(P<0.01).But the difference between the PCNA ASON and VEGF group was not significant(P>0.7). Conclusion PCNA ASON and VEGF gene transductionn are effective in preventing restenosis after balloon angioplasty caused vessel injury in rabbits.
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Objective To investigate the effect of NR2B antisense oligonucleotide on naloxone-induced withdrawal responses in morphine-dependent rats. Methods Famale SD rats weighing 230-270 g were anesthetized with intraperitoneal pentobarhital 60 mg/kg. Intrathecal (IT)catheter was placed at L3,4 interspace.Thirty-two rats in which FT catheter was successfully placed were randomly divided into 4 groups ( n = 8 each) : group C control; group MD morphine dependence; group AO NR2B antisense oligonucleotide (aNR2B) and group SO NR2B sense oligonucleotide (sNR2B) . In group MD, AO, SO chronic morphine dependence was induced by increasing doses of subcutaneous morphine for 6 days. The initial dose of morphine was 10 mg/kg twice a day and was increased by 10 mg/kg twice every other day and reached 50 mg/kg on the 6th day. In group AO and SO IT aNR2B or sNR2B 15 nmol was administered simultaneously with subcutaneous morphine. Morphine withdrawal responses was induced by IT naloxone 4 mg/kg and scored based on the responses (0 = normal; higher scores signify severer responses) . The weight loss was calculated.The expression of NR1, NR2A and NR2B mRNA in hippocampus was determined by RT-PCR. Results The morphine withdrawal syndrome and weight loss were significantly incresed in group MD, AO and SO, while NR2B mRNA expression in hippocampus was up-regulated in group MD and SO compared with group C. The morphine withdrawal syndrome and weight loss were significantly decreased, NR2A mRNA expression in hippocampus was up-regulated and NR2B mRNA expression was down-regulated in group AO compared with group MD. There was no significant difference in NR1 mRNA expression between the 4 groups . Conclusion NR2B antisense oligonucleotide can suppress morphine withdrawal responses through the regulation of NMDA receptor level and construction in hippocampus.