目的 探讨反义ATM多核苷酸（AS-Lipo）作用于裸鼠移植瘤对喉鳞状细胞癌(Hep-2)放射治疗敏感性的影响。 方法 用克隆生存率来检测喉鳞状细胞癌放射治疗后的生存能力；用流式细胞仪来分析喉癌细胞的凋亡情况；用Tunel染色法检测各组瘤体中细胞凋亡情况。 结果 在同等剂量的放射治疗下，AS-Lipo组的生存分数最低。接受4 Gy放射治疗后，行流式细胞仪检测，结果发现AS-Lipo组的凋亡率为（30.7 ± 1.31）%，远高于转染Sen-Lipo、Mis-Lipo、Lipo组。ATM AS-Lipo+放疗组的细胞凋亡数显著高于单独放射治疗组；ATM AS-Lipo+放射治疗组裸鼠瘤体内的凋亡指数为（17.12 ± 4.2）%，与其他组比较差异有统计学意义。 结论 反义ATM多核苷酸增强了裸鼠喉鳞状细胞癌对放射治疗的敏感性。
Objective To investigate the reversal effect of antisense phosphorothioate oligonucleotide （ASOND) on human hepatoma resistant cells. Methods Human hepatoma resistant cells SMMC-7721 was transfected with synthetic antisense phosphorothioate oligonucleotide complementary to the 5′ region flanking the AUG initiation codon mediated by lipofectamine. In vitro drug sensitivity was measured by MTT assay. The expression of P-170 was determined by flow cytometry and mRNA was assessed by RT-PCR. Results ASOND inhibited the expression of mRNA and p-170 in SMMC-7721, enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug. The best inhibitory effect was achived by the dose of 0.5μmol/L. Conclusion ASOND enhanced the sensitivity of SMMC-7721 to chemotherapeutic drug and reversed the multidrug resistance of SMMC-7721 partially.
ObjectiveTo explore the diagnostic value of ultrasound elastography (USE) combined with long non-coding RNA actin filament associated protein 1 anti-sense RNA 1 (AFAP1-AS1) mRNA in thyroid fine-needle aspiration (FNA) wash-out fluid for distinguishing benign from malignant thyroid nodules. MethodsThe patients with thyroid nodules who were treated in the Shenzhen Futian District Second People’s Hospital from January 2020 to June 2022 were collected. Before operation, the patients’ thyroid nodules were evaluated by the USE score and the AFAP1-AS1 mRNA in the thyroid FNA wash-out fluid was detected. The pathological result of the thyroid nodule after operation was as a gold standard for diagnosis of malignant thyroid nodules. The clinical diagnostic value of USE score combined with AFAP1-AS1 mRNA in the FNA wash-out fluid of the benign and malignant thyroid nodules were analyzed. ResultsA total of 174 thyroid nodules (124 patients) were detected in this study, of which 62 (45 patients) were histologically diagnosed as malignant. There was a statistical difference in the comparison of the composition ratio of USE score grading between the benign and malignant thyroid nodules (Z=8.82, P<0.001). The point of USE of the benign thyroid nodules was statistically lower than that of the malignant thyroid nodules [2.28±1.16 vs. 4.26±1.01, mean difference (MD) and 95% confidence interval (95%CI)=2.98 (2.76, 3.20), t=30.85, P<0.001]. The AFAP1-AS1 mRNA in the FNA wash-out fluid of the malignant thyroid nodules was statistically higher than that of the benign thyroid nodules [1.45±0.27 vs. 1.13±0.16, MD (95%CI)=1.45(1.39, 1.50), t=10.69, P<0.001]. Pearson correlation analysis showed that there was a positive correlation between the USE score of thyroid nodules and the expression of AFAP1-AS1 mRNA in the FNA wash-out fluid (r=0.58, P<0.001). The sensitivity and specificity of USE score in combination with expression of AFAP1-AS1 mRNA in the FNA wash-out fluid for diagnosing the malignant thyroid nodules by receiver operating characteristic (ROC) curve was 93.5% and 88.4% respectively. The area under the ROC curve (95%CI) was 0.91 (0.86, 0.96). Conclusion According to preliminary results of this study, USE score combined with AFAP1-AS1 mRNA in the thyroid FNA wash-out fluid is more sensitive and shows a potential diagnostic performance than USE score or AFAP1-AS1 mRNA detection alone for distinguishing benign from malignant thyroid nodules.
Objective To investigate the effect on expression of c-myc and proliferating cell nuclear antigen （PCNA） of vein grafts transferred by c-myc antisense oligodeoxynucleotides（ODN） of soluble stent. Methods A rabbit model of common carotid arteries grafted by external jugular veins was constructed in 50 New Zealand rabbits and were randomly divided into five groups, 10 rabbits each group. Control group： no stents ; group 1： soluble stent ; group 2： soluble stent with sense-ODN; group 3： soluble stent with antisense-ODN; group 4.. soluble stent with mismatch-ODN. At 7 d, 28 d and 90 d after surgery, vein grafts were harvested. The expression of c-myc and PCNA were identified by immunochemistry methods. Results At 7d, 28d, 90d after surgery, the expression of c-myc and PCNA of the intima and media of vein grafts in control group, group 1, group 2, group 4 were higher significantly than that in group 3 （P〈0. 01）. At 28d, 90d after surgery, the expression of c-myc in five groups were higher than that in the same group at 7d after surgery （P〈0. 01）. Conclusion Soluble stent can transfer ODN effectively. C- myc antisense-ODN transferred by soluble stent can inhibit significantly the expression of c-myc and PCNA in the intima and media of vein grafts.
Objective To investigate an inhibitive effect of the chitosan nanoparticles with the proliferation cell nuclear antigen （PCNA）-antisense oligo deoxy nucleotides (ASODN) on the intimal cell proliferation after the vein grafting.Methods Fiftyfour male SD rats, weighing 450-600g, were randomly divided in the experimental group and the control group of 27 rats each. In the experimental group, the chitosan nanoparticles with PCNAASODN were infused into the anastomosis segment of the right jugular artery and vein; then, the anastomosis segment was transplanted to the jugular artery on the same side. The rats in the control group were infused with normal saline by the same procedures. There were 24 rats in each group which used to experiment. The hemodynamic data were obtained from the Doppler ultrasound examinations at 1, 2, 3 and 4 weeks. The specimens were taken. Immunohistochemistry, Westernblot, and bloodvesselwall histopathology were performed at the different week points. Results There was no significant difference in the thrombogenesis rate between the experimental group and the control group (3/27 vs. 3/27,P＞0.05). During the 4 week observation, PCNA Westernblot showed that the PCNA level was lower in the grafted vein and the anastomosis segment in the experimental group than in the control group. The indexes of the PCNA postive proliferating cells in the intimal area (0.13%±0.11%，0.79%±0.28%，0.45%±0.29%, 0.43%±0.25%) and the medial area (1.90%± 0.84%，2.11%±0.98%，2.48%±0.77%，2.17%±0.36%) were significantlydecreased at 1,2,3 and 4 weeks in the experimental group when compared with those in the control group（P＜0.05）. The lumen areas in the grafted vein (88.71±16.96，95.98±21.44，88.48±32.81，97.86±34.11 μm 2) and the anastomosis segment (41.49±3.34，45.15±11.65，46.27±8.90，51.62±8.85 μm 2) were significantly greater in the experimental group than in the control group (P＜0.05). The ratios of the initmal area to the medial area in the grafted vein (22.73%±3.11%，32.40%±4.55%，45.14%±3.19%，45.70%±5.01%) and the anastomsis segment (41.49%±3.34%，45.15%±11.65%，46.27%±890%，51.62%±8.85%) were significantly smaller in the experimental group than in the control group（P＜0.05）. The maximum velocities (Vmax) of the blood flow inthe grafted vein and the anastomsis segment were almost the same in the two groups at 1 week, but had different changes at the next 3 weekpoints. In the control group, the Vmax of the blood flow gradually increased and at 3 weeks it reached the peak point; however, at 4 weeks it decreased. In the experimental group,the Vmax of the blood flow gradually decreased, and at 3 weeks it decreased to the lowest point; however, at 4 weeks it increased. So, at 4 weeks the Vmax of the blood flow in the grafted vein and the anastomsis segment was almost the samein the two groups. There was no significant difference in the Vmax of the bloodflow between the two groups (P＞0.05), but in the same group there wasa significant difference at the different time points. Conclusion The chitosan nanoparticles with PCNAASODN can effectively inhibit the intimal cell proliferation after the grafting of the blood vessel, so that the neointimal thickening can be prevented.
Objective To investigate the expression and clinical value of long chain non-coding RNA nicotinamide nucleotide hydrogenase antisense RNA1 (LncRNA NNT-AS1), motor neuron and pancreas homeobox protein 1 antisense RNA1 (MNX1-AS1) in lung cancer patients. Methods This study selected 128 patients diagnosed with lung cancer admitted to The Third Medical Center of the General Hospital of the People’s Liberation Army from April 2020 to April 2021 as a cancer group. During the same period, 128 patients with benign pulmonary nodules were regarded as a benign group, and 128 healthy individuals who underwent physical examination were selected as a control group. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect the levels of LncRNA NNT-AS1 and MNX1-AS1 in serum. A three-year follow-up was conducted on all lung cancer patients, with 52 patients in the death group and 76 patients in the survival group. Receiver operator characteristic (ROC) curve was applied to analyze the diagnostic value of serum LncRNA NNT-AS1 and MNX1-AS1 for the occurrence of lung cancer and their predictive value for prognosis. Results Compared with the control group, the serum levels of LncRNA NNT-AS1 and MNX1-AS1 were obviously increased in the benign group and the cancer group (P<0.05). Compared with the benign group, the levels of LncRNA NNT-AS1 and MNX1-AS1 in serum of the cancer patients were obviously increased (P<0.05). The area under ROC curve (AUC) of serum LncRNA NNT-AS1 combined with MNX1-AS1 for the diagnosis of lung cancer was higher than that of LncRNA NNT-AS1 and MNX1-AS1 alone (ZLncRNA NNT-AS1~LncRNA NNT-AS1+MNX1-AS1=2.496, P=0.013; ZMNX1-AS1~LncRNA NNT-AS1+MNX1-AS1=2.831, P=0.007). The levels of LncRNA NNT-AS1 and MNX1-AS1 were related to tumor differentiation, clinical stage, and lymph node metastasis (P<0.05). Compared with the survival group, the serum levels of LncRNA NNT-AS1 and MNX1-AS1 in the death group were obviously increased (P<0.05). The AUC of combined prediction for lung cancer prognosis by serum LncRNA NNT-AS1 and MNX1-AS1 was higher than that predicted by LncRNA NNT-AS1 and MNX1-AS1 alone (ZLncRNA NNT-AS1~LncRNA NNT-AS1+MNX1-AS1=2.539, P=0.011; ZMNX1-AS1~LncRNA NNT-AS1+MNX1-AS1=3.377, P=0.001). Conclusion LncRNA NNT-AS1 and MNX1-AS1 are highly expressed in serum of lung cancer patients, and both have certain value in diagnosis and prognosis evaluation of lung cancer.
Objective To evaluate the effect of vascular endothelial cell growth factor (VEGF) antisense oligodeoxynucleotides (ASODNs) on the expression of VEGF in rats with oxygen-induced retinopathy. Methods Thirty newborn Sprague-Dawley (SD) rats were randomly divided into 3 groups:normal control group, disposal group and non-disposed group, The animal models with oxygen-induced proliferative retinopathy were established by raising the rats in hyperoxic environment. Retrobulbar injection was performed with VEGF ASODNs or normal saline on the rats in 3 groups respectively. The intraocular tissues (all the tissues except the cornea, sclera, and lens) and serum were collected, and the expressions of VEGF were determined by using competitive enzyme immunoassay.Results The expressions of VEGF in intraocular tissues of rats in disposal group were significantly lower than those in non-disposed group (P＜0.05), and there was no statistical difference between the disposal and normal control group (P＞0.05). There was no significant difference of the expressions of VEGF in serum of rats between the disposal and non-disposed group (P＞0.05), which were both lower than those in the normal control group (P＜0.05). Conclusion VEGF ASODNs could significantly inhibit the expression of VEGF in intraocular tissues. (Chin J Ocul Fundus Dis，2003，19：172-174)
Objective To investigate the effect of phosphorothioate antisense oligonucleotides(AS-ODN) on suppressing multidrug resistance-associated protein gene(MRP) in human drug-resistant hepatocellular carcinoma cell line (SMMC-7721/ADM). Methods Cell line was transfected with a synthetic S-ODN complementary to the coding region of MRP mRNA, Lipofectamine acting as carrier. The drug sensitivity was measured by MTT assay. The expression of MRP mRNA was detected by RT-PCR and the expression of P190 was detected by flow cytometry. Results AS-ODN inhibited expression of MRP mRNA and P190 and promoted sensitivity to daunorubicinum and adriamycinum. Conclusion AS-ODN can reduce the expression of MRP gene. MDR caused by MRP is an important cause of multidrug resistance of SMMC-7721/ADM.
Objective To investigate the inhibitory effect of survivin antisense oligonucleotides (ASODN) on proliferation of pancreatic cancer cells PANC-1. Methods The ASODN and sense oligodeoxynucleotides (SODN) were complementary to survivin sequences. FAM-marked ASODN was transfected into PANC-1 cells mediated by positive ion liposome as ASODN group. Blank control group (normal cells), negative control group (normal medium)， and SODN group were established for comparison. The transfection efficiency was detected by flow cytometry (FCM) after transfection; MTT assay was used to detect cytotoxicity; Cell morphological changes were examined by transmission electron microscopy; The cell cycle and apoptotic rate were analyzed by FCM; Immunohistochemical staining techniques were used, and the expressions of survivin were observed under light microscopy, examined and analysed by computer image. Results ①The transfection efficiency was 31.9%, 37.4%, 41.4%, 52.6%, 24.2%, 11.4%, 16.1%, and 15.5% when the transfecting concentration of ASODN was 50, 100, 150, 200, 250, 400, 600, and 800 nmol/L, respectively; The transfection efficiency was 12.0%, 50.8%, and 11.2% when the inoculated cells was 2×104/well, 2×105/well, and 2×106/well, respectively; The transfection efficiency was 58.8%, 34.0%, and 23.6% when 2 μl, 3 μl, and 4 μl liposome was used during transfection, respectively. ②Cell gap was oversize, morphous was round, adherent cells were less after transfection under fluorescence microscope. ③The inhibition rate in the ASODN group was higher than that in each control group (Plt;0.05) on 24, 36, 48 h after treating by survivin ASODN, which increased as time prolonged (Plt;0.05). ④The apoptosis showed a ladder-shaped line in the ASODN group. ⑤Apoptotic morphology was demonstrated in the ASODN group, such as apoptotic cells with nuclear chromatin highly concentrated, crescent nuclear staining aggregated by the side nuclear membrane, nucleolus disappeared by AO and EB stains. ⑥The apoptotic rate 〔（38.1±3.4）%〕 in the ASODN group was higher than that in the SODN group 〔（4.16±1.7）%〕, Plt;0.05. ⑦G2/M cell cycle arrested in the ASODN group. ⑧After transfection, the expression of survivin protein in the ASODN group was significantly lower than that of each control group (Plt;0.05). Conclusions The optimal transfection conditions are as following: the cell count of 2×105/well, concentration of ASODN 200 nmol/L, and cationic liposome oligofectamine 2 μl, respectively. Survivin ASODN can inhibit the proliferation of pancreatic cancer cells and induce their apoptosis.