| Objective: Bone marrow suppression is one of the most common adverse reactions in malignant tumor chemotherapy, peripheral blood leucocyte reduce as the main performance, which can cause the body function decline, resistance to abate, secondary infection such as serious adverse reactions.According to our previous researches acupuncture bone marrow inhibition of basis, this study and influence in the development of bone marrow hematopoietic cells of hematopoietic stem/progenitor cell survival, proliferation and differentiation of the direction of Notch signaling pathway as the breakthrough point, discusses the Notch signaling pathway on genetic variations associated with acupuncture and moxibustion improve adverse reaction after chemotherapy, protect bone marrow hematopoietic cells and improve bone marrow inhibition, increase peripheral blood leukocyte related pathways and molecular biology mechanisms;Acupuncture and moxibustion in improving bone marrow suppression, enhance leukocyte function link of differences.At the same time the research can also be used for acupuncture to treat other diseases haematology and hematopoietic injury to provide new treatment idea and laboratory basis.Methods: Kun Ming(KM) mice,SPF, male, 18±2g, 40 mice. In the temperature of 20o~25oC, relative humidity 60% free diet three days in the experimental environment of the implanted tumor.The stage in an aseptic operation, put the required items in sterile operation stage uv light irradiation for 30 min.Extraction of saline 13.8 ml in the triangle flask.Left hand hold wedding mice, the right hand of mice after abdominal iodine volts first alcohol disinfection, with 1 ml syringe needle(5ml) extraction ascites 2.3ml, slow injection Erlenmeyer flask containing 13.8 ml of normal saline, blending, gently reached 7 times diluent.In 1 ml syringe needle(5ml) diluent extracted cells 1 ml, left hand holding the rat, right hand first to 40 mice left armpit disinfection(alcohol) after iodine volts first, and then with a syringe underarm injections, in the process of needle stab into the subcutaneous first, and then slowly to the left armpit direction, reach inside armpit again slowly injected with tumor cells 5ml×106/0.2 diluent, then push the needle while rotating the needle to 90° out needle, pressing with sterile swabs pinhole for a few seconds at the same time, avoid liquid leakage, finally let go of the mouse,with its tail in the cage,complete plant tumor.Vaccination in 7 days, blood check total number of WBC, choose weight 22+2g, 32 mice,tumors had 0.8cm in diameter, close to the basic WBC, were randomly divided into the tumor-burdened blank group, the tumor-burdened model group, the tumor-burdened acupuncture group, the tumor-burdened moxibustion group and each group of 8 mice;Numbered points cage and picric acid staining.The tumor-burdened model group, the tumor-burdened acupuncture group and the tumor-burdened moxibustion group of mice celiac injection of cyclophosphamide(CTX)150mg/kg, 4h after drug withdrawal model successed, namely the tumor-burdened blank group mice abdominal cavity inject equal dose of normal saline.Each group of the tumor-burdened mice each choose one pair on the self-made wood treated in turn.The tumor-burdened blank group, accompanied by the tumor-burdened model group treatment;and The tumor-burdened acupuncture group of mice selected acupoints "Da Zhui", "Ge Shu"(double), "Shen Shu"(double), "Zu San Li"(double) Jia Jian(Made in Wu Xi of Jiang Shu province) brand beauty filiform needle(specification:0.19cm×10mm, needle handle 20mm), Used the pipe into a straight stitch, into the needle depth of 3mm, retaining needle 6mins. once a day,five days;The tumor-burdened moxibustion group of mice also selected these acupoints "Da Zhui", "Ge Shu"(double), "Shen Shu"(double), "Zu San Li"(double),used special beauty moxa(specification: 0.4cm×25cm) lighting, fixed at 2cm distance acupoint skin every hole suspended moxibustion 3 min, once a day, five days in a row. The tumor-burdened blank group, the tumor-burdened model group synchronization is fixed, but no treatment.From until the tumor-burdened mice CTX injection to treat after 5 days, every day in the morning time, according to the numbering sequence cut each mouse tail vein blood, optical microscope to detect peripheral blood leukocyte count, treatment on the second day after 5 days in dry ice tumor-burdened bilateral femur and tibia in mice.Groups at the early application of small flux gene expression profile chip preliminary screening groups of normal mice bone marrow hematopoietic cells genetic variations associated with Notch signal pathway;Because on the one hand the tumor-burdened mice and normal mice for the same batch, just with tumors had different, on the other hand due to funding, therefore,on the basis of the preliminary screening,using immunohistochemical method to further screening of different groups tumor-burdened bone marrow hematopoietic cells in mice genetic variations associated with Notch signal pathway.Realtime PCR method analysis of different groups tumor-burdened bone marrow hematopoietic cells in mice genetic variations associated with Notch signal pathway m RNA change;Western Blot method to verify different groups tumor-burdened bone marrow hematopoietic cells in mice Notch signaling pathways genetic differences associated with the amount of protein expression.Interpretation of the Notch signaling pathway is bone marrow suppression, peripheral blood leukocyte reduction in acupuncture combination such as key mechanism.Results:(1) "Experiment 1st" results show, according to the results of the tumor-burdened mice from 2 days after the injection of CTX peripheral white blood cells that is reduced. Compare to the tumor-burdened blank group, after treatment 3 days the tumor-burdened model group, the tumor-burdened acupuncture group, the tumor-burdened moxibustion group of white blood cell count fell to the lowest(P<0.05), 4 days after treatment began to rebound, at this time the tumor-burdened acupuncture group, the tumor-burdened moxibustion group of white blood cell count is the tumor-burdened model group picks up.5 days after treatment, the tumor-burdened model group, the tumor-burdened acupuncture group, the tumor-burdened moxibustion group, all white blood cell count is close to a basic level of white blood cell, and compare to the tumor-burdened model group, the tumor-burdened acupuncture group and the tumor-burdened moxibustion group of white blood cell count is higher than but there was no statistically significant difference(P>0.05).(2) "Experiment 2nd" results show, according to the results of preliminary screening through small flux gene expression profile chip, in the tumor-burdened group, the tumor-burdened blank model group, the tumor-burdened acupuncture group, the tumor-burdened moxibustion group of mice bone marrow hematopoietic cells on the Notch signal X pathway, gene were screened seven differences: notch1, notch2, jag1, jag2, delta1, numb1, numb2.(3) "Experiment 3rd" results show, Used RT-PCR method to the tumor-burdened bone marrow cells in mice Notch signaling pathways jag1, notch2, significant differences in the genes numb1, m RNA transcription protein profile of numb2 test. Compare to the tumor-burdened blank group, the tumor-burdened model group jag1, elevated notch2 protein expression levels, but there was no statistically significant difference(P>0.05), reduced numb1, numb2 protein expression levels, and numb2 has significant difference(P<0.05); Compare to the tumor-burdened model group, the tumor-burdened acupuncture group, the tumor-burdened jag1 moxibustion group and notch2 protein expression levels were elevated, numb1 is reduced, but there were no statistically significant difference(P>0.05); Compare to the tumor-burdened model group, the numb2 protein expression levels of the tumor-burdened moxibustion group rise, there was significant difference(P<0.05); Compare to the tumor-burdened acupuncture group, the tumor-burdened moxibustion group numb2 protein expression levels higher than the tumor-burdened acupuncture group, but there was no statistically significant difference(P>0.05).(4) "Experiment 4th" results show, Used RT-PCR method to the tumor-burdened bone marrow cells in mice Notch signaling pathways jag1, notch2, significant differences in the genes numb1, m RNA transcription protein profile of numb2 test. Compare to the tumor-burdened blank group, the tumor-burdened model group jag1, elevated notch2 protein expression levels, but there was no statistically significant difference(P>0.05), reduced numb1, numb2 protein expression levels, and numb2 has significant difference(P<0.05); Compare to the tumor-burdened model group, the tumor-burdened acupuncture group, the tumor-burdened jag1 moxibustion group and notch2 protein expression levels were elevated, numb1 is reduced, but there were no statistically significant difference(P>0.05); Compare to the tumor-burdened model group, the numb2 protein expression levels of the tumor-burdened moxibustion group rise, there was significant difference(P<0.05); Compare to the tumor-burdened acupuncture group, the tumor-burdened moxibustion group numb2 protein expression levels was higher than the tumor-burdened acupuncture group, but there was no statistically significant difference(P>0.05).(5) "Experiment 5th" results show, Used Western blot method to the tumor-burdened bone marrow cells in mice Notch signaling pathways jag1, notch2, significant differences in the genes numb1, protein expression profile of numb2 test. Compare to the tumor-burdened blank group, the tumor-burdened model group jag1, elevated notch2 protein expression levels, and have significant difference(P<0.05), numb1, numb2 protein expression levels were low, but there was no statistically significant difference(P>0.05); Compare to the tumor-burdened model group, the tumor-burdened acupuncture group, the tumor-burdened jag1, notch2 protein expression levels of moxibustion group were lower, and it has significant difference(P<0.05), numb1, numb2 protein expression content increases, but there was no statistically significant difference(P>0.05); Compare to the tumor-burdened acupuncture group, the tumor-burdened jag1, notch2 protein expression levels of moxibustion group were lower, numb1, numb2 protein expression levels rise, although no statistical significance difference(P>0.05), but acupuncture was better than that of moxibustion.Conclusion:(1) The base of white blood cells in the tumor-burdened mice was a little higher than the normal mice.(2) Acupuncture and moxibustion can relieve CTX tumor-burdened mice bone marrow suppression caused by chemotherapy, increase peripheral blood leukocytes, its curative effect in line with previous findings mentor group, after treatment of 3 days to the minimum, 4 days began to rebound, 5 days to normal, and the tumor-burdened acupuncture group and the tumor-burdened moxibustion group of white blood cell count was higher than the tumor-burdened model group, acupuncture and moxibustion, there was no significant difference between the two methods, but acupuncture is better than that of moxibustion.(3) Through preliminary screening of gene expression profile chip, small flux in the tumor-burdened blank group, the tumor-burdened model group, the tumor-burdened acupuncture group, the tumor-burdened moxibustion group of mice bone marrow hematopoietic cells on the Notch signal pathway, were screened notch1, notch2, jag1, jag2, delta1, numb1, numb2 seven different genes.By the immunohistochemical method of small flux gene expression profile chip preliminary screening of the tumor-burdened bone marrow hematopoietic cells in mice Notch signaling pathways seven differences further gene screening, to determine jag1, notch2, numb1, numb2 gene has significant difference.(4) Based on the above index variation and correlation analysis, interpretation of acupuncture and moxibustion to protect hematopoietic stem cells, reduce bone marrow suppression, promote blood and bone marrow hematopoietic cells, Notch signaling pathways related to distribution and the relationship between the expression of gene and protein, it is a key mechanism may be: bone marrow suppression caused by chemotherapy with CTX, acupuncture and moxibustion can increase the tumor-burdened Numb protein on the Notch signal pathway in mouse bone marrow tissue protein(numb1, numb2) expression, at the same time cut notch2, jagged1 protein expression, thereby inhibiting excessive activation of Notch signaling pathways, relief from bone marrow suppression caused by chemotherapy of CTX.Verify the "Notch signaling pathways is acupuncture and moxibustion against the key of the combined action of bone marrow suppression, improve hematopoietic mechanism" of the hypothesis. |
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