| Leukemia, which affects the people's living quality seriously, is the haematological malignancy with very high morbidity rate and malignant degree. Since 80's in 20 centuries, the treatment of the leukemia have made rapid progress, high dose chemotherapy, the hematopoietic stem cell transplantation and the symphysis application of the immunization therapy, have raised the survival rate of the leukemia patients. But high-dose chemotherapy has the serious poisonous side effect, as to the hematopoietic stem cell transplantation, high expensive cost, and seriously complications such as graft vs host disease limits its clinical extensive application. Therefore, investigating novel, valid treatment method with little poisonous side effect, become the focus concern of the basic and clinical researcher of blood disease.The theoretical basis of the anti-leukemia of antisense technique is based of the alkali repair principle, which make use of the antisense oligonucleotides to terminates the proto-oncogene and the oncogene, making leukemia cell to divide toward maturity or inducing the cell apoptosis, so as to treating leukemia. It has been already confirmed that the oncogene c-myc and human telomerase reverse transcriptase (hTERT) gene are expressed in the myelogenic leukemia cell line HL-60 cells. The two genes probably are relevant with occurrence of leukemia. The oncogene c-myc is an important member of the myc family, its product belongs to the phosphatase protein of nucleoprotein, which play important role in the cell proliferation,differenation and the apoptosis process. A great deal of research showed that the c-myc gene excessive expression is closely related to various tumors. Recent data have manifested that the c-myc may participate in the activate of hTERT and telomerase. It has been already confirmed that the telomerase enzyme activity increase play an important role in the process of the malignant tumor formation. The telomere is the protection structure that locates in the chromosome bitter end of eukaryotic cells, the telomerase can synthesize the telomere, complement the telomere that shortened by the cell division, making the cell immortally lived. The hTERT is the part of telomerase enzyme activity, activating the important function during the process of activation of telomerase.Leukemia is polygenes, polystages, polysteps occurrence process with complicated mechanism, Blocking the expression of one gene alone, usually can't obstruct the occurrence of the tumor completely. It is still in the study that whether combination of two antisense oligodeoxynucleotides (ASODN) with different effect will improve the inhibition to the tumor. It is not clear what changes that the hTERT and c-myc gene have in the process of leukemia, what effect on the proliferation, differentiation networks, and what influence on the telomerase enzyme activity and the cell apoptosis so far. The Cisplatin (CDDP), a kind of alkylating agent and a very important chemotherapy agent within various chemotherapy scheme, can cross linkage with DNA molecular which can result in DNA hurt after entering the cell, having the good anti-tumor effect. Because of its toxicity function of the hematopoiesis stem cell, Cisplatin is limited in the treatment of leukemia. The side effect of ASODN is little, the specificity is strong and it is unknown whether it can reduce the dosage and toxicity of Cisplatin. According to above-mentioned consider, in this experiment, the hTERT and c-myc ASODN were packaged with liposome and were transfected into HL-60 cells, closing the expression of hTERT and c-myc gene, proliferation, apoptosis and the telomerase enzyme activity were observed in the HL-60 cells. It is very interesting to investigate the effects and mechanism of ASODN of different leukemia gene.This experiment, which is the first time in China, transfected the hTERTASODN into the HL-60 cells, investigating the effects on the neoplasm of HL-60 cells in the animal as well as the inhibition of growth of HL-60 cells. The effects of the two ASODN in combination with Cisplatin on the cell proliferation, apoptosis were also investigated in this study. So this study was to extend the indications of ASODN and provide experimental basis for the therapy to tumor. Methods:1. MTT and trypan blue exclusion method were adopted to investigate the proliferation of the HL-60 cells after the transfection of hTERT and c-myc ASODN. Growth appearance and morphology of the cells were observed with microscope.2. RT-PCR method was adopted to examine the expression of the genes of HL-60 cells which were treated with hTERT and c-myc ASODN.3. The polyacrylamide gel electrophoresis of TRAP-PCR and ELISA were adopted to examine the influence of ASODN on the telomerase enzyme activity of the HL-60 cells.4. DNA and the cell cycle (cell apoptosis rate) were analyzed with flow cytometer, DNA ladder and TUNEL technique were used to detected the apoptosis of the cells.5. 15 Balb/c female nude mice with HL-60 cells were randomly distributed to A, B, C group, which were transfused with hTERT ASODN, sense oligodeoxynucleotides (SODN) and control, neoplasm formation were investigated in the nude mice. The tumor physical volumes were calculated every 7 days.6. Neoplasm were cultured to 10-15th generation and then were transfused into the nude mice, after the neoplasm came out, 0.25 ml of 2xl02nmol/ml hTERT ASODN were transfused to the rats body everyday. After 8 days, pathologic slice and TUNEL were adopt to observe the effects of hTERT ASODN on the cell proliferation, apoptosis.7. After 48hs of the transfection of hTERT and c-myc ASODN in the cells, The effects of 2.5 and 5umol/L of Cisplatin, the combination of Cisplatin and ASODN were observed respectively. Trypan blue stain method was adopted to investigatethe proliferation of the HL-60 cells. Flow Cytometry and TUNEL were used to detect the apoptotic cells. Morphology and apoptotic body of the cells were observed with microscope.8. SPSS 11.5 statistical software was applied to statistics the data. To categorical variable the chi-square test was used to analyze. To numerical variable the datawas expressed with jc±s, ANOVA was used to compare these data Thesignificant level was a =0.05. Results:1. MTT and trypan blue stain: 72hs after the transfer with 5, 10, 20, 30umol/L of hTERT and c-myc ASODN, the HL-60 cells count and the SDH enzyme activity is obviously inhibited. Compared with the SODN and the control group, the inhibition effect of ASODN were significant different (PO.01); There were not significant different (P>0.05) among different concentration groups of SODN but ASODN(P<0.01); Effects of the combination group of hTERT and c-myc ASODN was not significant different compared with the hTERT and c-myc group respectively (f>0.05). The inhibition effects on the HL-60 cells of 20umol/L of ASODN were not different with 30umol/L of ASODN. The inhibition effects of 20|xmol/L of ASODN after24, 48, 72, 96hs were significantly different compared with the SODN and the control group (PO.01). The effect was not different between the 72,96hs.2. RT-PCR: 72hs after 5, 10, 20umol/L of hTERT or/and c-myc ASODN were transfected to HL-60 cell, expression of gene mRNA decreased, and the effect of 20|x mol/ L ASODN was much more obviously.3. TRAP-PCR: 24,48, 72hs after the cells were treated with 10,20umol/L of hTERT or/and c-myc ASODN, telomerase enzyme activity decreased compared with that of the SODN group. There was a higher effect of hTERT ASODN than the c-myc ASODN group. TRAP-ELISA: 72hs after the transfection, the inhibition effect of the ASODN were significant different comparing with the SODN and the control group (P<0.05). There were significant different among different concentrationgroups of ASODN (P<0.01); Effects of hTERT ASODN group was significant compared with c-myc ASODN group and combination group respectively (P<0.05). The effects of hTERT ASODN is the most powerful, whose 50% inhibition concentration ( IC50) was 20umol/L. 24, 72, 120hs after 20umol/L of ASODN transfected, telomerase enzyme activity were detected, the inhibition effects of the ASODN were significant different compared with the SODN and the control group (PO.05).4. With Flow cytometer, there were earlier apoptotic spike when the HL-60 cells were treated with 20umol/L of hTERT or/and c-myc ASODN at 72hs, the apoptosis rate were 25.20^3.38%, 21.44±2.56%s, 22.65±3.02% respectively. With agarose gel electrophoresis, many apoptotic bands could be found when the HL-60 cells were treated with ASODN, and no apoptotic bands was found when the cells were treated with hTERT SODN or the control. With TUNEL, it was found that 20umol/L of ASODN had significant apoptotic induction effect when the HL-60 cells were treated for 48, 72, 96hs, and the positive apoptotic cells increased with the time, Which were significantly different compared with the SODN and the control group (PO.01).5. Neoplasm formation experiment showed that neoplasm formed after the treatment of hTERT ASODN after 16-17d (A group), while hTERT SODN and the control (B,C group) after 12-13d. Neoplasm formation rate was 3/5 in A group and 4/5 in B,C group, the physical volume of average tumor were smaller in A group than in B,C group (/><0.01).The growth of the cells in A group were obviously suppressed when the cells were stained with the HE.6. The animal experiment in vivo showed: after 8 days of treatment, the nude mice were killed by pulling the cervicis and the tumor tissue were made to slice and were detected by pathology, HE stain and the TUNEL. The result 5 could be seen with HE slice. Typical cell apoptotic morphology character could be seen with microscope. With TUNEL method, a great deal of positive cells could be found in the tumor tissue, while few positive cells in the control tumor tissue.7. Trypan blue exclusion showed: After 48hs of treatment with hTERT, c-mycASODN, CDDP was added for more 24hs, significant inhibition effect on the HL-60 cells could be found, which were significantly different compared with the groups of SODN in combination with CDDP or the CDDP alone(PO.Ol). There were much more apoptotic cells and typical cell apoptotic morphology character could be seen with cell morphology observation. With TUNEL, much more apoptotic cells could be found, which were significantly different with the groups of SODN in combination with CDDP or the CDDP alone, the control (PO.01). The same results were found with Flow Cytometer (.PO.01). Conclusions1. hTERT and c-myc ASODN had significant inhibition effects on the proliferation of the HL-60 cells and expression of purpose genes mRNA. The inhibition effects were sequence specificity and density, time-depended.2. hTERT and c-myc ASODN had significant inhibition effects on the telomerase enzyme activity. The inhibition effects of hTERT ASODN were stronger.3. It was confirmed that hTERT and c-myc ASODN had the apoptosis induction effects on HL-60 cells.4. The combination of hTERT and c-myc ASODN did not change much to the inhibition effects on the proliferation of the HL-60 cells.5. Nude mice neoplasm model of HL-60 cells were set up in this experiment. It was confirmed that hTERT ASODN could inhibit the formation of neoplasm.6. There was a more powerful neoplasm formation ability with the neoplasm tissue, and hTERT ASODN could inhibit the growth of the tumor and induce the tumor cell apoptosis.7. The combination of hTERT and c-myc ASODN with CDDP could increase the inhibition effects of CDDP on the proliferation of the HL-60 cells, as well as the cell apoptosis induction effects.
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