| Objective: Chemotherapy as one of the main means of treating the malignant tumor, One of its greatest adverse reactions that led to bone marrow and bone marrow microenvironment damage caused by bone marrow suppression, peripheral blood leukocyte reduced, leading to the body followed by a series of adverse reactions. Bone marrow hematopoietic microenvironment settlement 、 proliferation 、 differentiation 、 development 、 maturity of hematopoietic parenchymal cells plays an important role in regulating. In this study, acupuncture around the repair and the regulation of cyclophosphamide(CTX) after chemotherapy bone marrow microenvironment,Bone marrow stromal cells(BMSC) is an important component of hematopoietic microenvironment,cell adhesion molecules by producing secretion,cell growth factors and extracellular matrix components and regulating and play a role.In this study,on the theme of the Bone marrow hematopoietic microenvironment in bone marrow hematopoietic microenvironment of adhesion molecules and hematopoietic factors as the breakthrough point,Using flow cytometry to detect the bone marrow stromal cells(BMSC) cell cycle change in the bonemarrow hematopoietic microenvironment;Using light microscope observation,the granulocyte and red blood cell series bone marrow pathological morphology;And the use of RT-q PCR and Western-blot method detection of gene and protein expression of adhesion molecules VCAM-1、ICAM-1 and hematopoietic factor SCF、TGF-β1 in bone marrow hematopoietic microenvironment. In order to investigate the study of acupuncture on chemotherapy bone marrow microenvironment of hematopoietic growth factors and adhesion molecules as well as the regulation of repair and protective effect on bone marrow hematopoietic microenvironment, acupuncture after chemotherapy to improve bone marrow microenvironment injury, enhance white blood cells, providing experimental basis.Methods:Selection SPF male Kunming(KM) mice 120, weight 18 ± 2g, at a temperature of 20 ℃ ~25 ℃, relative humidity of about 60% clean grade laboratory, the free feeding for 3 days, to make the model. After weighing each mouse sequence, using stratified random grouping method, the mice were divided into groups 1d, 3d group, 5d groups of 40, then turn the 1d group, 3d group, 5d mice successively points for the control group, model group, acupuncture group, moxibustion group n = 10.Specific modeling method according to the People’s Republic of China Ministry of Health, Bureau of Drug Administration compiled the "new drug(medicine) compilation of pre-clinical research guidelines," the use of cyclophosphamide(CTX) established by intraperitoneal injection of bone marrow suppression model, according to 100 mg / kg / d dose, continuous application of 3 days. Based pharmacokinetic cyclophosphamide(CTX), four hours after administration of drugs lose activity, namely the establishment of a successful model(specific modeling basis: Zhongshan Medical College, eds basic pharmacology People Health Press, 1979: 442).Blank group based 0.02 ml / g of dosage, intraperitoneal injection of saline. Model four hours after successful treatment. Blank group, model group, accompanied by fixed, without any treatment. Acupuncture group first secured to the homemade mouse mouse board, use a special needle Hua card beauty, concrete specifications: 0.19 × 10 mm, needle handle length 20 mm, followed by acupuncture mouse Ojo, diaphragm acupoints, Shenshu, the full three years, the needle 3mm, needle 6min, daily treatment 1, wherein the treatment of 1 1d mice, 3d mice for 3 times, 5d mice for 5 times. The mice were fixed in the moxibustion group homemade mouse board, use a special beauty thin moxa, moxa specifications: 0.4 × 25 cm, after the mouse ignited moxa moxibustion Ojo, Geshu Shenshu, Zusanli, from the skin of mice height 2cm, per hole 3min, treatment day 1, wherein the treatment of 1 1d mice, 3d mice for 3 times, 5d treatment group were five times.1d Group, 3d Group, after the end of the 5d group therapy, observe the effects of acupuncture on the peripheral blood leukocytes of law, and by light microscopy of bone marrow biopsy in bone marrow erythroid, myeloid lines morphological changes, and then the application flow cytometry was observed in mice bone marrow microenvironment changes of cell cycle of bone marrow stromal cells(BMSC), then apply the RT-q PCR and Western-Blot method to detect bone marrow microenvironment adhesion molecule VCAM-1, ICAM-1 and hematopoietic factors SCF, gene and protein expression of TGF-β1. CTX chemotherapy through the observation of acupuncture and moxibustion in mice bone marrow microenvironment and the impact of changes in adhesion molecules of hematopoietic growth factors, chemotherapy explore acupuncture improve the protection and repair of bone marrow from the bone marrow microenvironment micro environment, improve the mechanism of peripheral blood leukocytes. Experiments based on the strict implementation of the whole process promulgated by the National Science and Technology "guidance for the Care of Laboratory Animals" in the relevant regulations.Results:1. "experiment 1st " results show that Compared with the control group, 1d model groups 、acupuncture and moxibustion groups decreased the peripheral blood cells, Compared with the control group, the other groups were lower, there was a significant difference(P <0.05). 5d group acupuncture group, moxibustion group were elevated peripheral white blood cells of mice, the normal value has been reached.2. "experiment 2nd" results show that the mice were observed by the light microscopy 1d group, 3d group, 5d group of Bone marrow Wright- Giemsa staining biopsy, The results showed that with Compared with model group,acupuncture group and moxibustion group to increase the number of red blood cell system, increase the number of cell lines tablets, an increase in the number of nucleated cells lines, reduce fat cells, increasing the sinuses. As the duration of treatment, the change is more obvious.3. "Experiment 3rd" results showed that Application of flow cytometry group 1d, group 3d,group 5d mice bone marrow microenvironment stromal cells(BMSC) cell cycle G1 phase, S phase, G2 phase cells percentage change, The results showed that Compared with the control group, model group 3d group(P <0.05) and 5d group(P <0.01) percentage of cells in G1 phase BMSC decreased, the difference was statistically significant; Acupuncture group, 1d group(P <0.05) percentage of cells in G1 phase BMSC significantly increased, the difference was statistically significant; compared with the control group, model group 1d group(P <0.05) and group 3d, 5d group( P <0.01) the percentage of cells in s phase BMSC significantly increased, the difference was statistically significant; compared with the control group, model group 1d group, 3d group(P <0.05) and 5d group(P <0.01) BMSC of G2 percentage of cells was significantly increased, the difference was statistically significant; Compared with the model group,acupuncture group and moxibustion group can reduce the CTX chemotherapy in mouse bone marrow cells in the hematopoietic microenvironment of bone marrow stromal cells in the G1 phase, in which the 3d group, 5d groups were statistically significant, P <0.05. Compared with the model group, acupuncture and moxibustion can improve 3d group of cells in the S phase BMSC, acupuncture and moxibustion can reduce the 5d group of cells in the S phase BMSC are statistically different, P<0.05. Compared with the model group, acupuncture and moxibustion can increase group 1d, group 3d, group 5d BMSC of cells in the G2 phase, all have significant difference, P <0.05. Compared with the acupuncture group, moxibustion group 5d group(P <0.05) BMSC G1 phase, S phase cell percentage was significantly increased, the difference was statistically significant. Compared with acupuncture, moxibustion 3d group(P <0.05) percentage of cells in G2 BMSC was significantly increased, the difference was statistically significant.4. "Experiment 4th" results show Using RT-q PCR method for group 1d, group 3d, group 5d CTX chemotherapy of mice bone marrow microenvironment adhesion molecule VCAM-1, ICAM-1 and hematopoietic factors SCF, TGF-β1 gene were detected. The results show that: in the 1d set of experiments, compared with the control group, model group VCAM-1, SCF m RNA was down-regulated, with significant differences with statistical significance(P<0.05), ICAM-1, TGF-β1 m RNA expression increased, but no significant difference(P>0.05), compared with the model group, acupuncture group, moxibustion group were not statistically significant, but after acupuncture, moxibustion VCAM-1, SCFm RNA expression was up-regulated, ICAM- 1, m RNA expression of TGF-β1 were down, but there was no significant difference(P> 0.05).In 3d set of experiments, compared with the control group, model group VCAM-1, SCF m RNA was down-regulated, with significant differences with statistical significance(P<0.05), ICAM-1, m RNA expression of TGF-β1 increased, but there was no significant difference(P>0.05). Compared with the model group, acupuncture group and moxibustion group VCAM-1, SCFm RNA expression was up-regulated, acupuncture group had significant differences with statistical significance(P<0.05), moxibustion group had no significant difference(P>0.05); While the acupuncture group and moxibustion group, ICAM-1, m RNA expression of TGF-β1 were lower, but not statistically significant(P> 0.05).In 5d set of experiments, compared with the control group, model group VCAM-1, SCF m RNA was down-regulated, with significant differences with statistical significance(P<0.05), ICAM-1, m RNA expression of TGF-β1 increased, significant difference was statistically significant(P<0.05). Compared with the model group, acupuncture group and moxibustion group VCAM-1, SCFm RNA expression was up-regulated, there was no significant difference(P>0.05); While the acupuncture group and moxibustion group, ICAM-1, m RNA expression of TGF-β1 were down acupuncture group had significant differences with statistical significance(P <0.05), moxibustion group was not statistically significant(P> 0.05).5. " Experiment 5th " results showed that using Western-Blot method 1d Group, 3d Group,5d group CTX chemotherapy in mouse bone marrow hematopoietic microenvironment adhesion molecule VCAM-1, ICAM-1 and hematopoietic growth factors SCF, TGF-β1 protein amounts detected.The results show that: in the 1d set of experiments, compared with the control group, model group VCAM-1, SCF protein content decreased, and were statistically significant(P<0.05). Table compared with the model group, acupuncture group, VCAM-1, SCF protein increased, and VCAM-1 protein has a significant difference was statistically significant(P<0.05); the acupuncture group ICAM-1, TGF-β1 protein content decreased, and TGF-β1 protein significant difference was statistically significant(P<0.05); acupuncture for 1 day, compared with the model group, moxibustion group, VCAM-1, SCF protein increased, and VCAM-1 protein has significant difference was statistically significant(P <0.05); moxibustion group ICAM-1, TGF-β1 protein content decreased, and have a significant difference was statistically significant(P <0.05). Compared with acupuncture, moxibustion VCAM-1, SCF protein levels, ICAM-1, TGF-β1 protein content decreased, no significant difference(P> 0.05).In 3d set of experiments, compared with the control group, model group VCAM-1, SCF protein content decreased, and VCAM-1 has significant difference was statistically significant(P<0.05); ICAM-1, TGF-β1 protein content increased, with significant difference was statistically significant(P<0.05); compared with the model group, acupuncture group VCAM-1, SCF protein content increased, and there is a significant difference, statistically significant(P<0.05); acupuncture group, ICAM-1, TGF-β1 protein content decreased, no significant difference. Acupuncture treatment for 3 days, compared with the model group, moxibustion group VCAM-1, SCF protein content increased, and VCAM-1 protein has a significant difference was statistically significant(P<0.05); moxibustion group ICAM-1, TGF-β1 protein content decreased with significant difference was statistically significant(P<0.05). Compared with acupuncture, moxibustion VCAM-1, SCF protein content increased, ICAM-1, TGF-β1 protein content decreased, no significant difference was not statistically significant(P> 0.05).In 5d set of experiments, compared with the control group, model group VCAM-1, SCF protein content decreased, and a significant difference was statistically significant(P<0.05); ICAM-1, TGF-β1 protein content increased, significant difference was statistically significant(P <0.05); compared with the model group, acupuncture group VCAM-1, SCF protein content increased, and SCF protein significant difference was statistically significant(P<0.05);acupuncture group, ICAM-1, TGF-β1 protein content decreased, and TGF-β1 protein significant difference was statistically significant(P<0.05). Acupuncture treatment for 5 days, compared with the model group, moxibustion group, VCAM-1, SCF protein content increased, and a significant difference was statistically significant(P<0.05); moxibustion group ICAM-1, TGF-β1 protein content decreased, with TGF-β1 protein significant difference was statistically significant(P<0.05). Compared with acupuncture, moxibustion VCAM-1, SCF protein content increased, ICAM-1, TGF-β1 protein content decreased. Compared with acupuncture, moxibustion VCAM-1, SCF protein content increased, ICAM-1, TGF-β1 protein content decreased, no significant difference was not statistically significant(P> 0.05).Conclusion:1. Acupuncture and moxibustion can relieve cyclophosphamide(CTX) chemotherapy in mouse bone marrow suppression, to enhance the role of the number of peripheral blood leukocytes, 3 days group began to rise, 5 days group to reach normal value.2. Acupuncture and moxibustion can affect the bone marrow morphological changes, can promote red blood cell system, there is the number of cells increased cell nucleus, it can reduce the number of leukocytes.3. Alkylating agents CTX destroyed the Cycle of bone marrow stromal cells to produce bone marrow suppression, seriously affecting the bone marrow function, acupuncture and moxibustion can promote cell repair after CTX chemotherapy, bone marrow stromal cells, accelerate marrow from G1 to S phase of transformation, reinforced synthetic bone marrow stromal cell DNA, extended G2 phase, while using cell G2 arrest time, DNA repair as soon as possible, accelerate cell mitosis into proliferation, increase of bone marrow stromal stromal cells the ability of cells to repair and protect the bone marrow microenvironment and promote reconstruction of bone marrow function.4. Acupuncture mitigate damage bone marrow microenvironment cyclophosphamide(CTX) chemotherapy-induced, and its mechanism may be: Acupuncture can increase bone marrow microenvironment adhesion molecule VCAM-1 and hematopoietic SCF positive regulation factor gene and protein expression, can reduced bone marrow microenvironment and hematopoietic adhesion molecules ICAM-1 gene and protein expression of negative regulatory factor TGF-β1, and play a protective role in the bone marrow microenvironment.5. Bone marrow microenvironment by adhesion molecules and hematopoietic cytokines, may be acupuncture restoration and protection of bone marrow microenvironment after chemotherapy damage, enhance white blood cells, the main pathway to promote recovery of immune function. |
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