Effect of Dexamethasone and Aquaporin-1 Antisense Oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

Obstructive Effects of Ultrasonic Microbubble Intensifier on CHG-5 Cell with Survivin Antisense Oligonucleotides Transfection

Objective： To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides （ASODN） transfection. Methods： Antisense oligonucleotides targeting survivin mRNA was designed and synthesized. Four regimen groups were designed, group A： survivin antisense oligonucleotides transfected with ultrasonic microbubble intensifier combined with ultrasound irradiation, group B： survivin antisense oligonucleotides transfected with lipofectamine combined with ultrasound irradiation, group C： survivin antisense oligonucelotides with lipofectamine transfection, group D： blank control. The expression changes of surviving protein were measured by immunohistochemical staining and Western blotting, and MTT assay was used to measure the changes of proliferation. Results： Survivin protein expression in group A was decreased significantly in human glioma cell line CHG-5 than other groups（P〈0.05）, and the proliferating rate of CHG-5 in group A was also significantly inhibited（P〈0.05）. Conclusion： Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively.

Enhanced therapeutic effects of combined chemotherapeutic drugs and midkine antisense oligonucleotides for hepatocellular carcinoma

AIM: To evaluate the effect of combined antisense oligonucleotides targeting midkine (MK-AS) and chemotherapeutic drugs [cisplatin(DDP), 5-fluorouracil (5-FU) and adriamycin (ADM)] on inhibition of HepG2 cell proliferation, and to analyze the efficacy of MK-AS used in combined ADM in in situ human hepatocellular carcinoma (HCC) model. METHODS: HepG2 cells were treated with MK-AS and/or chemotherapeutic drugs mediated by Lipofectin, and cell growth activity was determined by MTS assay. An in situ HCC model was used in this experiment. MK- AS, ADM and MK-AS + ADM were given intravenously for 20 d, respectively. The animal body weight and their tumor weight were measured to assess the effect of the combined therapy in vivo. RESULTS: Combined treatment with MK-AS reduced the IC50 of DDP, 5-FU and ADM in HepG2 cells. MK-AS significantly increased the inhibition rate of DDP, 5-FU and ADM. Additionally, synergism (Q 1.15) occurred at a lower concentration of ADM, 5-FU and DDP with combined MK-AS. Combined treatment with MK-AS and ADM resulted in the more growth inhibition on in situ human HCC model compared with treatment with chemotherapeutic drugs alone. CONCLUSION: MK-AS increases the chemosensitivity in HepG2 cells and in situ human HCC model, and thecombination of MK-AS and ADM has a much better in vitro and in vivo synergism.

Combination of telomerase antisense oligonucleotides simultaneously targeting hTR and hTERT produces synergism of inhibition of telomerase activity and growth in human colon cancer cell line

AIM: To investigate synergism of inhibition of telomerase activity and proliferation of human colon cancer cells by combination of telomerase antisense oligonucleotides (ASODNs) simultaneously targeting human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) in vitro. METHODS: ASODN of hTR and ASODN of hTERT were transfected into human colon cancer SW480 cells by liposomal transfection reagents. Telomerase activity of SW480 cells was examined using telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay (PCR-ELISA). Proliferation activity of SW480 cells was tested by methyl thiazolyl tetrazolium assay. Apoptosis and cell cycle were analyzed by flow cytometry. RESULTS: The telomerase activity and cell survival rate in SW480 cells transfected with 0.2 μmol/L of ASODN of hTR or ASODN of hTERT for 24-72 h were significantly decreased in a time-dependent manner compared with those after treatment with sense oligonucleotides and untreated (telomerase activity: 24 h, 73%, 74% vs99%, 98%; 48 h, 61%, 55% vs98%, 99%; 72 h, 41%, 37% vs 99%, 97%; P<0.01; cell survival rate: 24 h, 88%, 86% vs594%, 98%; 48 h, 49%, 47% vs94%, 97%; 72 h, 44%, 42% vs92%, 96%; P<0.01). Moreover, the telomerase activity and the cell survival rate in SW480 cells treated by the combination of telomerase anti-hTR and anti-hTERT were more significantly suppressed than single anti-hTR or anti-hTERT (telomerase activity: 24 h, 59% vs 73%, 74%; 48 h, 43% vs61%, 55%; 72 h, 18% vs41%, 37%; P<0.01; cell survival rate: 24 h, 64% vs88%, 86%; 48 h, 37% vs49%, 47%; 72 h, 25% vs44%, 42%; P<0.01). Meanwhile, the apoptosis rates in the combination group were markedly increased compared with those in the single group (24 h, 18.0% vs7.2%, 7.4%; 48 h, 23.0% vs13.0%, 14.0%; 72 h, 28.6% vs 13.2%, 13.75; P<0.01). Cells in combination group were arrested at G0/G1 phase. CONCLUSION: Telomerase anti-hRT and anti-hTERT suppress telomerase activity, and inhibit growth of human colon cancer cells probably via induction of apoptosis and retardation of cell cycle. Additionally, combined use of telomerase ASODNs targeting both hTR and hTERT yields synergistic action selective for human colon cancer.

Inhibition of PCNA Antisense Oligonucleotides Mediated by Liposome on mRNA Expression and Proliferation of h-RPE Cells

Summary： The proliferating cell nuclear antigen （PCNA） gene expression was blocked and retinal pigment epithelium （RPE） proliferation was inhibited by using antisense oligonucleotides （AS-ODN） mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy （PVR）. RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN （0.07, 0.28 and 1.12 μ mol/L and sense oligonucleotides （S-ODN with the same concentrations as AS-ODN） mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by ^3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression ofPCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density （AOD） was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN （0.28 and 1.12 μmol/L） markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant （P〈0.05 and P〈0.01, repectively） as compared with blank control group. AOD was well correlated with CPM （r=0.975）. It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.

Following Inhibition of BCL-2 by Antisense Oligonucleotides Compensatory Suppression of Apoptosis Involves the Direct Signal Transduction Pathway of LNCaP Cells

Previously we have shown that when LNCaP cells are treated with antisense oligonucleotides (oligos) directed against BCL-2, compensatory changes in non-targeted genes take place in attempts to restore apoptosis and promote tumor aggressiveness. In addition to the inhibition of BCL-2, we find that the apoptosis promoter caspase-3 activity is suppressed, the transcription activity of STAT-3 is enhanced, while other regulators (bax, clusterin, AKT-1) associated with mitochondrial regulated apoptosis and caspase cascade are either unchanged or undetectable. We now evaluate proteins associated with the second pathway of apoptosis activation mediated by direct signal transduction involving fas, fas-ligand (a tumor necrosis factor-like cell surface receptor aka CD95), as well as the similar programmed death cell surface receptor (PD-1) and its respective ligand (PD-1L). This study evaluates the growth inhibition of in vitro propagating LNCaP cells employing mono- and bispecific oligos directed against BCL-2 [the second binding site was directed against the epidermal growth factor receptor (EGFR)];and employing RT-PCR. The expression of these four proteins was evaluated. Expression of fas-ligand, PD-1 and PD-L1 were all significantly enhanced, whereas fas itself was undetectable. This suggests that in addition to pathways associated with the mitochondrial pathway of apoptosis, compensatory changes occur in the direct signal transduction pathway of this process. In addition to alterations in androgen sensitivity, growth factor expression and oncogene expression, these data suggest that suppressive BCL-2 therapy involves multiple pathways, including those involved with immune targeting and cytotoxicity and must be taken into account to make gene therapy more efficacious.

SELECTION AND THEIR ANTITUMOR ACTIVITY OF ANTISENSE OLIGONUCLEOTIDES TARGETING MESSENGER RNA OF VASCULAR ENDOTHELIAL GROWTH FACTOR RECEPTOR 2

Objective： To select the antisense oligonucleotides （asONs） which hybridize with the mRNA of vascular endothelial growth factor receptor2 （VEGFR2, also named as kinase insert domain-containing receptor：KDR） in an effective and specific way, and to investigate their antitumor activity in MCF-7 cells. Methods： The effective antisense oligonucleotides were chosen by computer prediction combined with oligonucleotide microarrays. The inhibition effect on MCF-7 cells proliferation was measured by MTT; and VEGFR2 expression was surveyed by Western-blotting and RT- PCR. Results： Using predicting secondary structure of VEGFR2 mRNA with RNA folding program, computer prediction designed 30 antisense oligonucleotide probes that were directed to local loose regions of RNA structure. In 30 probes, 4（4/30, 13.33%） antisense oligonucleotides showed strong hybridization intensities in oligonucleotide microarrays test and were selected. All these antisense oligonucleotides targeting 4 different sites of VEGFR2 mRNA lowered the level of VEGFR2 mRNA and protein present in MCF-7 cells. Proliferation of MCF-7 cells was reduced by 4 antisense oligonucleotides, respectively, in which asON1 was the most effective, with the inhibitory rates being 53.06% at 0.8 I.tmol/L. Conclusion： Combination of computer prediction with oligonucleotide microarrays is an effective way in selecting optimal antisense oligonucleotides. The antisense oligonucleotides showed good correlation between their antitumor activity and the hybridization intensities. The antisense oligonucleotides targeting VEGFR2 mRNA demonstrated prominent antitumor role in vitro.

P53 Regulation of Leukemia Cells with the Blockage of MDM2 by Antisense Oligonucleotides

Summary： The changes of expression and function of MDM2 and P53 by MDM2 specific antisense oligonucleotides were investigated in HL60 cells. Cells were divided into control group, AS group （MDM2 specific antisense oligonucleotides group）, cisplatin group, and combined treatment group. FCM analysis and Western blot and RT-PCR were used to estimate apoptosis and the expression of MDM2 and P53. Our results showed that the transfection of MDM2 specific antisense oligonucleotides obviously inhibited MDM2 expression （P〈0.01） and increased the expression of P53 （P〈0.05）. Apoptosis rate were reduced by MDM2 specific antisense oligonucletides and cisplatin （P〈0.01）. It is concluded that MDM2 specific antisense oligonucletides can inhibit the expression of MDM2, induce the expression of P53 and increase the apoptosis of leukemia cells after chemotherapy.

Effects of human papillomavirus type-specific antisense oligonucleotides on the proliferation of cervical cancer cells containing papillomavirus type 16

Objective: To investigate the effects of antisense phosphorothioate oligonucleotides (S--ODNs ) on theproliferation of cervical cancer cell line SiHa, which harbor HPV16. Methods: Three different antisenseoligodeoxynucleotides were used in this study. Two of them, S--ODN1 (AE6 ) and S--ODN2 (AE7 ), arecomplementary to sequences flanking the start codons of HPV16 E6 and E7 open reading frames,respectively. SODN3 is a nonsense sequence. SiHa cells were treated with various concentrations of oligos. Growth assay and 3HTdR incorporation were used. Results: Both AE6 and AE7 markedly inhibited the proliferation and DNA synthesisof SiHa cells which harbor HPV16 but had little effects on HeLa cells that do not. S--ODN3 had none of theseeffects. Conclusion: Antisense oligodeoxynucleotides can inhibit the proliferation and DNA synthesis of HPV16positive human cervical cancer cells.

Antisense therapy:a potential breakthrough in the treatment of neurodegenerative diseases

Neurodegenerative diseases are a group of disorders characterized by the progressive degeneration of neurons in the central or peripheral nervous system.Currently,there is no cure for neurodegenerative diseases and this means a heavy burden for patients and the health system worldwide.Therefore,it is necessary to find new therapeutic approaches,and antisense therapies offer this possibility,having the great advantage of not modifying cellular genome and potentially being safer.Many preclinical and clinical studies aim to test the safety and effectiveness of antisense therapies in the treatment of neurodegenerative diseases.The objective of this review is to summarize the recent advances in the development of these new technologies to treat the most common neurodegenerative diseases,with a focus on those antisense therapies that have already received the approval of the U.S.Food and Drug Administration.

Effects of connective tissue growth factor antisense oligonucleotides on the proliferation and collagen synthesis of the cultured human keloid fibroblasts in vitro

Objective: To explore the effects of connective tissue growth factor (CTGF) on the pathogenesis of human keloid. Methods: CTGF antisense oligonucleotides (ASODN) conjugated with isothiocyananate fluorescence was encapsulated by liposome, and then added into the human keloid fibroblasts (HKFs) culture media. The intracellular distribution of CTGF ASODN was observed by fluorescence microscopy in the fixed HKFs. The proliferation of HKFs was measured by MTT test. The collagen synthesis of HKFs was measured by 3H-proline incorporation method. Results: Compared with control group, the CTGF ASODN can inhibit the proliferation and collagen synthesis of the HKFs (P<0.01). Conclusion: CTGF ASODN has anti-fibrotic effects on keloid in vitro, and CTGF play an important role in promoting the fibrosis of keloid.

Study on Apoptosis-Inducing Effect of XIAP Antisense Oligonucleotides on Glioblastoma Cells in Vitro

OBJECTIVE To investigate the apoptosis-inducing effect of XIAP antisense oligonucleotides on glioblastoma cells in vitro. METHODS There were 4 groups in our experiment. Group A, as a cell control group, had normal cell culture and no treatment applied. Group B, as a blank control group, had normal cell culture and no liposome control of ASODN. Group C was N-ODN. Group D was the ASODN group. RT-PCR and Western blot assay were conducted to detect the expression of XIAP in all A-172 ceil groups after treatment with XIAP antisense oligonucleotides （ASODN）. MTT assay and flow-cytometry （FCM） detection were used to detect the ability of cell anchoring growth and apoptotic rates of all groups. The processing time was 72 h. RESULTS The expression of XIAP in the A-172 cells was greatly down-regulated, after treated with XIAP-ASODN. Among different concentrations of ASODN, the 300nM was the most optimal one. The down-regulation of XIAP obviously inhibited the succinate dehydrogenase （SDH） activity of the A-172 cells and the increased apoptotic rate of A-172 cells （87.45%） was significantly higher than that of the A-172 in the control groups. There was a statistically significant difference between the treatment and control groups （P 〈 0.01）. CONCLUSION The XIAP-ASODN can effectively regulate the expression of the XIAP down, as a result, inhibit the growth of the glioblastoma cells （A-172） and obviously increase the apoptotic rate of the A-172 cells. The results killing role of XIAP-ASODN to the of the study manifest an overt glioblastoma cells.

Depressive Effect of the Antisense Oligonucleotides of C-myc and PCNA on the Proliferation of VSMC

Objectives To study the depressive effect of the antisense oligonuceotides （ASODN） of c-myc and proliferating cell nuclear antigen （PCNA） on the proliferation of VSMC. Methods Taking the VSMC obtained from rat aorta tho- racalis cultured 4 - 8 generation as research object. The objects were divided into three groups to carry out control study： control group, PCNA ASODN group and c-myc ASODN group. The ASODNs＇ working concentration all were 1 ： 50. The depressive effect of ASODN on VSMC proliferation was investigated by cell counting, MTT and ^3H-TdR incorporation assay; PCNA and c-myc expression were detected by immunohistochemical method after transferring PCNA and c-myc ASODN into VSMC. Results ① PCNA and c-myc ASODN could inhibit the proliferation of VSMC significantly, compared with control group （ P 〈 0. 05）. ②Transferring PCNA and c-myc ASODN into VSMC obtained successfully ; the corresponding gene was inhibited obviously ; compared with control group （ P 〈 0. 05 ）. Conclusions PCNA and c-myc might play a considerable role in the VSMC proliferation process. The corresponding gene could be depressed successfully after transferring PCNA and c-myc ASODN into VSMC, and then the proliferation of VSMC was slowed down. This study presented a beneficial proposal and theoretical fundament for atherosclerotic treatment.

Effects of antisense oligonucleotides on theexpression of macrophage migration inhibitoryfactor on macrophages

To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with the sequences of antisense, 5′-TACGGATACAAGTAGCAC-3′; Sense, 5′-ATGCCTATGTTCATCGTG-3′; Missense, 5′-CTCTCAGACTCGATCTGT-3′. These phosphorothioate oligonucleotides were then transfected into cultured macrophages ( RAW264.7 ) by luciferase vector, and the transfected macrophages were incubated with Lipopolysaccharide (LPS) (1?ng/ml) for various periods of times and collected afterwards. The content of MIF protein in the cultural supernatants was determined by ELISA, cellular RNA extracted and the expression of MIF mRNA was examined by RT-PCR analysis. The experimental results showed that LPS could induce a time-dependent specific expression of MIF on macrophages, in which the MIF mRNA in cells and the MIF protein in cultural supernatants appeared after 3 h and reached their highest concentration at 9-12?h after LPS stimulation. The levels of mRNA and proteins in the macrophages treated with antisense olignucleotides were decreased significantly after stimulation with LPS in comparison with that of stimulation with LPS alone or with that with LPS plus sense or missense oligonucleotides. There were no differences among those without LPS stimulation. It is concluded that macrophages stimulated with LPS express MIF, and the antisense olignucleotides of MIF inhibit the expression of MIF mRNA as well as the secretion of MIF proteins in macrophages.

MAG_3和DTPA用于^(99)Tc^m标记Survivin ASON的对比研究

目的 比较巯基乙酰基三甘氨酸 (MAG3)和二乙烯三胺五乙酸 (DTPA)对99Tcm 标记Sur vivin反义寡核苷酸 (SurvivinASON)的影响。方法 用DNA合成仪合成 18碱基单链SurvivinASON ,在5′末端经氨基修饰后 ,分别用S 乙酰基 N 羟基琥珀酰亚胺 MAG3(S acetyl NHS MAG3)和环DTPA酸酐作为螯合剂 ,进行99Tcm 标记 ,并对其标记率、放化纯、比活度、稳定性、细胞摄取率和细胞清除率进行比较。结果 99Tcm MAG3 ASON和99Tcm DTPA ASON的标记率分别为 (6 5 2 2± 6 87) %和 (5 0 5 6±5 4 5 ) % ;放化纯分别为 94 %和 95 % ;比活度分别为 1190和 86 0kBq μg。室温条件下 ,与生理盐水保温 4h后 ,其放化纯均 >90 % ;与正常人血清保温 4h后 ,2种标记寡核苷酸均未见明显血清蛋白质结合。99Tcm MAG3 ASON在肝癌细胞中的摄取率高于99Tcm DTPA ASON(P <0 0 1) ,但两者在肝癌细胞中的摄取均明显高于正常肝细胞 (P <0 0 1)。结论 采用S acetyl NHS MAG3和环DTPA酸酐为螯合剂 ,用99Tcm 标记SurvivinASON可行 ,前者螯合效果优于后者。

FasL及FasL ASODN在舌鳞癌细胞免疫逃逸中的作用

目的研究Fas配体(FasL)及FasL反义寡脱氧核苷酸(ASODN)在舌鳞癌细胞免疫逃逸中的作用。方法对人舌鳞癌细胞中FasL表达及功能进行检测,设计合成特异FasL ASODN,转染舌鳞癌细胞,封闭舌鳞癌细胞的FasL表达。通过流式细胞术、逆转录聚合酶链反应(RT-PCR)和四唑盐(MTT)等方法,观察基因-蛋白-细胞效应。结果通过RT-PCR检测提示在脂质体的量一定的情况下,在一定范围内,ASO的浓度越高,转染效率越高。MTT/FCM结果显示ASO对SCC-9细胞的增殖有明显的抑制作用(P<0.05)。FCM对细胞凋亡的检测结果显示反义链组与正义链组、空白对照组及脂质体组相比,总凋亡率有明显差异(P<0.05)。结论 Fas/FasL途径是口腔鳞癌细胞免疫逃逸的重要机制之一,可作为免疫治疗的新靶点。

用等位基因特异性寡核苷酸(ASO)-PCR快速检测抗多菌灵的油菜菌核病菌

通过3对兼并性引物扩增获得与S.sclerotiorum抗药性相关的β-微管蛋白基因,全长1 685 bp,包含4个内元,相应的编码447个氨基酸。该基因与其它6种线状真菌的β-微管蛋白基因相比,氨基酸序列同源性达95.78%～97.66%,但内元数目和大小不同。比较S.sclerotiorum敏感型和抗药性菌株的β-微管蛋白基因,发现该基因第198位氨基酸由谷氨酸突变为丙氨酸,从而导致田间抗药性的产生。为了快速、准确监测田间抗药性频率,根据S.sclerotiorumβ-微管蛋白基因的突变位点设计了2对等位基因特异性寡核苷酸(ASO),直接以菌核的基因组DNA为模板扩增,所需时间约为6 h,抗药性检出率为100%,与传统菌丝直径法的测定结果相比检测准确率为96%,而传统的检测方法至少需要1～2周。

^(99m)Tc-MAG_3-ASON的制备及其在荷乳腺癌裸鼠中的分布

目的:探讨99mTc标记的反义寡核苷酸(ASON)的制备及其在荷乳腺癌裸鼠中的分布。方法:一步法合成巯基乙酰三甘氨酰-N-羟基琥珀酰亚胺酯(NHS-MAG3),并与c-erbB2 mRNA互补的5’末端氨基修饰的15个碱基的ASON偶联;随后进行99mTc的标记,并用Sephadex G25分离纯化99mTc-MAG3-ASON,并评价其稳定性;最后检测其在荷乳腺癌BALB/c裸鼠体内的分布。结果:99mTc-MAG3-ASON平均标记率为70.6%;纯化后,在室温下放置4h,其放化纯度为94.3%;与人血清孵育后,其放化纯度为93.8%;与血浆蛋白结合率为11.8%。乳腺癌组织的摄取率2h达峰值(6.09%ID/g)。结论:以NHS-MAG3为螯合剂制备的99mTc-MAG3-ASON具有良好的稳定性,在乳腺癌部位高浓聚,可望用于肿瘤的显像诊断和治疗。

脂质体介导的hTERT PEI/ASODN缩合体对乳癌MCF-7细胞生长及hTERT表达的影响

目的:探讨脂质体介导的人端粒酶逆转录酶(hTERT)聚乙烯亚胺(PEI)/反义寡核苷酸(ASODN)缩合体对乳癌MCF-7细胞生长及hTERT表达的影响。方法:MCF-7细胞分为3组,分别转染ASODN、PEI/ASODN和脂质体PEI/ASODN,采用激光共聚焦扫描观察摄入情况。另取MCF-7细胞分为9组:空白对照组、ASODN组、SODN组、溶媒组、空白脂质体组、PEI/ASODN组、PEI/SODN组、脂质体-PEI/ASODN组和脂质体-PEI/SODN组。转染后不同时间,分别采用MTT法、RT-PCR及免疫细胞化学方法检测MCF-7细胞的生长情况、hTERT蛋白以及 mRNA的表达。结果:脂质体介导的hTERTPEI/ASODN缩合体可以进入细胞内。转染后24、48和72h,9组间光密度值相比,差异均有统计学意义(F=11.187、30.283和33.080,P均<0.001)。与空白对照组相比,PEI/ASODN组、PEI/SODN组及脂质体-PEI/ASODN组光密度值降低(P<0.05)。与空白对照组相比,转染48h后hTERT蛋白和hTERT mRNA表达降低。结论:脂质体介导的hTERTPEI/ASODN缩合体能够有效进入细胞内,抑制MCF-7细胞的生长,其作用可能与抑制细胞中hTERT mRNA及蛋白的表达有关。