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【关键词】 Radiation dose; Sarcoma 180; Apoptosis; Cell cycle; Bone marrow
Experimental studies have proved that low dose radiation (LDR) can increase the body’s immune function, which is manifested as increased reactivity of immunologically competent cells, antibody production and tumorkilling activity[1]. The immune indexes are also improved in the tumorbearing mice with LDR combined with chemotherapy[2], but few reports have shown the changes in the tumors and chemotherapysensitive marrows. We did a research on this issue, and the results are reported as follows.
1 MATERIALS AND METHODS
1.1 Experimental animals and grouping
Aseptic Kunming strain male mice, (18±22) grams, 4-6 weeks, provided by Drug Control Institution of Qingdao, were randomly divided into five groups: blank control group (BC group), direct tumorbearing group (DT group), LDR tumorbearing group (LDR group), CTX chemotherapy group (CTX group) and LDR tumorbearing with CTX chemotherapy group (LDR+CTX group). The mice were raised conventionally without limits of water and food.
1.2 Irradiation
FCC7000 isocentricity revolution 60Co therapeutic machine, made by Shandong Xinhua Medical Apparatus Factory, was used. The mice were put in a selfmade carton, with a water mould in the middle to filter radiation. The radiation distance was 209.5 cm, the dosage rate being 16.4 mGy/min, and the total dosage 75 mGy. All the mice were irradiated only once.
1.3 Implantation of tumor cells
S180 sarcoma cells, provided by Shandong Medical Institute, were passaged twice in the abdominal cavity of the mice. Suspension containing 6.5×107/mL cells in logarithmic growth phase was conventionally prepared and 6.5×106/mL cells were implanted subcutaneously in the left inguen of the mice.
1.4 Methods of irradiation
Five days after the implantation, maximum horizontal diameter a(cm) and vertical diameter b(cm) were measured twice respectively with slide gaud. The mice with tumors of either too large (ab>1.00 cm2) or too small (ab<0.20 cm2) sizes were abandoned. The average tumor sizes were calculated according to the following formula: V=(1/2)ab2［1］. Ifno difference existed in the sizes of tumors among all the groups, the mice of the LDR、LDR+CTX and DT groups were given 75 mGy γirradiation, and 36 hours (the 7th day) later 3 mg CTX (300 mg/kg) were injected into the abdominal cavity in both the CTX and LDR+CTX groups. We repeated the process on the 8th-11th days and measured the tumor diameters every two days.BC group do not need treatment. All the mice were sacrificed on the 12th day.
1.5 Observation items
Tumor growth: The tumors were exposed, and the average tumor sizes were directly measured and calculated with the method described above. Tumor cell apoptosis and cell cycle: First, a piece of the tumor tissue was taken, conserved in PBS fluid. Nest, tissue was grinded in 180 munylon net, flushed by PBS fluid and made into fresh single cell suspension mechanically. Then, it was centrifuged in 780 turns/minute to remove chips lasting 3 minutes,washed by 0.01 mol/L PBS fluid twice, poured superfluous fluid,added isvolumic protease which digested it for 30 minutes in 37 ℃, dyed by 200 μL PI for 30 minutes in 4 ℃. Last, tumor cells were collected by flow cytometry(BectonDikson FACS Vantage). The data were analyzed with ModFit 2.0 software. Bone marrow proliferation: Double thighbone of the mice (1 cm) were taken and the marrow cells were sucked out to be made into single cell suspension,which was typed by 200 μL PI for 30 minutes in 4 ℃. The cell cycle was detected by flow cytometry; And the proliferation index (PI) was calculated on the following formula: PI=(S+G2/M)/(G0/G1+S+G2/M)［4］.
1.6 Statistical processing
The data were shown by mean±standard deviation (x-±s). ttest was used to compare the results between two groups.
2 RESULTS
2.1 Suppression of tumor growth
Tumor volumes of the experimental groups were significantly different from those of the direct tumorbearing groups (P<0.05). Table 1.Table 1 Suppression of tumor growth (略)
2.2 Assay of tumor cell apoptosis and cell cycle
The apoptotic rate of mouse tumor cells and the number of S stage cells in LDR group increased more significantly than those in the direct tumorbearing group, while the number of G2/M stage cells decreased more significantly. Few apoptotic cells were observed in CTX group and LDR+CTX group, in which the number of G1 stage cells increased, but that of S stage cells decreased significantly. Table 2.Table 2 Changes of tumor cell apoptosis and cell cycle (略)
2.3 Proliferation of bone marrow
The concentration of marrow cells in the DC and LDR groups didn't differ significantly from that in the BC group, while that in the CTX and LDR+CTX groups decreased significantly. The number of G1/G0 and S stage cells in the CTX and LDR+CTX group decreased significantly compared with that of the BC, DT, and LDR group, while the number of G2/M stage cells in the CTX and LDR+CTX groups increased significantly. The proliferation index (IP) had also an significant increase. Table 3.
Table 3 Proliferation of mouse bone marrow (略)
3 DISCUSSION
It has been proved by a great deal of experimental studies and human body observations that LDR could induce adaptive response, enhance immune function, which is manifested in the increase of immunologically competent cell reactivity, antibody production, tumorkilling activity, interleukin2, and interferon quantity on mammalian and human body[5]. LDR could also decrease chromosome damage, gene mutation, cell death and tumor incidence. LDR differs from the routine high dose radiation in that its target might be substance out of the karyon such as cytoplasm, membrane of cell and some of conductive systems instead of on DNA. Some researchers in China contribute a lot by using LDR combined with radiotherapy, especially in the reduction of the side effects of radiation[6]. It has been shown that LDR combined with CTX reduces significantly the growth of the tumors of the tumorbearing mice, and the pulmonary metastases of Lewis cells. The simultaneous analysis of the immune items showed that LDR could improve the immune function of the tumorbearing mice depressed by chemotherapeutant and enhance some items such as the activity of NK and CTL cells, the phagocytic function of the macrophagocytes, and the reaction of IL2 to splenic cells[7]. Our previous experiment showed that[8],within 24 h after LDR, G1 phase arrest appeared in the tumor cells of the mice, and decreased significantly in S phase; but no change was observed in G2/M phase compared with the control group. 48 h later, G1 phase arrest of the tumor cells disappeared, cells and apoptosis in S phase increased while cells in G2/M phase decreased significantly against the control group. This experiment showed that LDR itself could induce apoptosis of a large proportion of tumor cells after 48 hours, with S phase cells increasing in a large proportion, but only for a short time. CTX could not induce significant tumor cells of mice apoptosis, but G1 phase arrest, S phase decrease, and the trend to synchronization of cell cycles. CTX effect on tumor cells increased after 36h following LDR, G1 phase was arrested more significantly, and cells proliferation decreased ulteriorly. Our experiment showed that tumor volumes of the LDR group, CTX group and LDR+CTX group decreased in turn, which indicates that the antitumor effect increased one by one, and testifies that LDR has a good adjuvant effect on chemotherapy.
One of the difficulties of tumor radiotherapy and chemotherapy lies in how to avoid the damage to the normal tissues caused by radiation and chemotherapy agents, how to reduce the secondary affection and germ plasm damage caused by severe suppression of immune system in conventional radiochemotherapy. Our experiment indicates that the concentration of marrow cells decreased and proliferous proportion of marrow cells increased with the subcutaneous transplantation of tumor cells and with CTX; While the concentration increased and the proportion decreased with LDR. If LDR was given 36 hours before the chemotherapy, the concentration of marrow cells in the LDR group increased 9 times as much as in the CTX group, and the proliferous proportion had a further increase, which indicates that low dose radiation could decrease significantly the damage to bone marrow caused by CTX.
【参考文献】
[1]YU H S, SONG A Q, LU Y D, et al. Effects of low dose radiation on tumor growth, erythrocyte immune function and SOD activity in tumorbearing mice[J]. Chin Med J, 2004,117(7):10361039.
[2]HORST H A, HORNY HP. Tumorinfiltrating lymphoreticular cell[J]. Cancer, 1991,68:23962402.
[3]ZHANG Y, LI X Y, LIU S Z. The enhancement effects of low dose radiation combined with cyclophosphamide on implantation tumor growth[J]. J Norman Bethune University Med sci, 1998,24:571573.
[4]JOHN L H. Principles of flow cytometry[J]. Cytometry, 1998,3(Suppl):79.
[5]LIU S Z. Threshold issue of radiation damage commemoration of the Xray discovery by Wilhelm Conrad Roentgen for 100 years[J]. Oncology Section Foreign Medical Sciences, 1995,24:571573.
[6]WANG X J, SU S J, YI H S. Effects of low dose radiation on tumor growth and radiotherapy in tumorbearing mice[J]. Chin J Radial Med Prot, 1996,16:157160.
[7]WOLFF S. Low dose exposure and the induction of adaptation[J]. Science Publishers, 1992:2128.
[8]LIU ZIMIN, YU HONGSHENG. Effect of low does radiation on tumor growth and hormesis in erythrocyte system in tumorbearing mice[J]. Acta Academiae Medicinae Qingdao Universitatis, 2007,43(1):4144.
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