ABSTRACT
Objective To observe anti-sense phosphorothioate oligonucletide (ASPSODN) targeted directly to hTERT mRNA to its inhibiting effect on aimed gene and the influence on the telomerase activity, cellular proliferation, cell apoptosis of K562 cells. Methods Human leukemia cell line K562 was transfected with anti-sense oligonucleotide ASPSODN by liposome. The proliferation activity of K562 cell line was determined by using methyl thiazolyl tetrazolium assay, and telomerase activity was detected by TRAP-PCR-ELISA. Flow cytometry was adopted to examine apoptotic rate and cell cycle. RT-PCR was used to detect the expression of target gene hTERT mRNA. Results 0.6 μmol/L ASPSODN (0.42 ±0.16) was remarkably decreased the expression of hTERT mRNA, Telomerase relative activation was decreased by 52 %. According to 0.6 (μmol/L ASPSODN caused significant the inhibition of K562 cell growth. Apoptotic rate and cell cycle was examined by 0.6 μmol/L ASPSODN with flow cytometry. The cell apoptosis rate of 0.6μmol/L ASPSODN were 10.31 %. It showed the cells treated with 0.6 uU PSASODN arrested in G_1/C_0. The ratio of cells in G_2/M and S period was reduced. But there was no characteristic apoptosis peak. Conclusion ASPSODN targeted hTERT can inhibit the expression of target gene hTERT mRNA, and decrease the telomerase activity of K562 cells. ASPSODN can inhibit strongly the proliferation of K562 cell and induce cell apoptosis by decreasing telomerase activity.
ABSTRACT
Objective To select an efficient method to inhibit telomerase activity, antisenseoligodeoxynucleotide and plasmid-vector mediated RNAi against hTERT were used to inhibit telomerase activity. The inhibiting effects of the two methods were compared. Methods Against hTERT mRNA, siRNA and oligodeoxynucleotide were designed and transfected into K562 cells by liposome. Effective and specific siRNA strands were selected and then plasmid was constructed and transfected into K562 cells; followed by analysis of the results. Results hTERT mRNA were detected after the three chemo-synthesized strands were transfected. It was found that si-hTERT-1 and si-hTERT-2 were effective, but si-hTERT-3 had no effect. The inhibiting effect of hTERT mRNA lasted only 48 h and disappeared at 72 h. Two siRNA strands were sieved and plasmids were constructed and transfected into K562 cells. In the P-1 group, hTERT mRNA was 0.39±0.13 at 48 h, 0.57±0.32 at 72 h. In the P-2 group, hTERTmRNA was 0.55±0.20 at 48 h, 0.88±0.23 at 72 h.In the P-1 group, the relative telomerase activity was 0.42±0.07 at 48 h, 0.31±0.08 at 72 h. In the P-2 group was 0.49±0.27 at 48 h, 0.39±0.03 at 72 h. The best concentration of siRNA was 100 μmol/L. The best concentration of ASODN was 0.6 μ mol/L. hTERTmRNA was 0.42±0.16 at 24 h, 0.71±0.18 at 48 h. Relative telomerase activity was 0.52±0.002 at 24 h, 0.482±0.018 at 48 h. Conclusion Both ASODN and RNAi targeting hTERT can inhibit the expression of hTERT mRNA, and then inhibit telomerase activity. The inhibiting effect is closely relative to the targeting site. The inhibiting effect of RNAi is better than that of ASODN. RNAi has better efficiency and lasts for a longer time. Plasmid mediated RNAi has better inhibiting effect than the chemo-synthesized siRNA.
ABSTRACT
Objective To promote the level of morphological diagnosis and estimate prognosis combining the result of bone marrow histotomy and the swear of 82 cases of malignant hemology disease. Methods We collected 82 cases of malignant hemology disease, extracted marrow by one step and routinly made bone marrow swear and dyeing, made histotomy by embedding plastic. Diagnosis of malignant hemology disease refered to the standard of WHO 2001. Results Bone marrow histotomy was superior to bone marrow swear in diagnosing MF and hypocellular MDS. Bone marrow histotomy was easier to find cell of lymphoma than bone marrow swear. Bone marrow histotomy was a more sensitive index of tumor load of MM.It was also helpful for judging acute change of CML and relapse of acute leukemia. Conclusion Level of diagnosing malignant hemology disease can be promoted efficiently by combining marrow histotomy with bone marrow swear