| BACKGROUNDBone metastasis is one of the common complications in patients with advanced cancer. Clinical on lung cancer, breast cancer, prostate cancer, and other solid tumors appear the most easily accounted for bone metastasis, bone metastasis85%[1]. Primary lung cancer with bone metastases of advanced lung cancer patients30-40%[2], mainly distributed in the ribs, spine, pelvis and limbs and other parts. The vast majority of osteolytic destruction, and few in bone destruction. Because bone and periosteum damage, often cause local pain, movement of bone and joint disorders, pathological fractures, hypercalcemia, which makes the survival of patients with decreased quality of life, the median survival time of primary lung cancer patients with bone metastasis is about8-10months [3]. At present in patients with bone metastasis is still no significant effective treatment scheme, the comprehensive treatment methods, including systemic chemotherapy, local radiotherapy, endocrine therapy and radioactive isotopes, is its effect on cancer cells. While the bone effect drug known, only two Lin acid salts for inhibiting osteoclast and play a role in the treatment, some scholars study of bisphosphonates can inhibit osteoclasts, intervention violated tumor cells to bone, bone injury caused by [4] reduced tumor. Currently used to promote bone formation drugs PTH (1-34) can promote bone formation, also can change the bone micro structure. Whether can use its promoting bone formation and bone destruction of bone metastasis cancer on delay, and delay the pathological fracture?Parathyroid hormone (Parathyroid, Hormone, PTH) is a kind of promoting bone formation drugs [5], FDA approval in20102002American, PTH (1-34) approval to enter the China. There have been numerous studies show that intermittent small dose injection of PTH (1-34) could significantly increase the bone density, improve bone microstructure, reduce fracture risk [6,7]. At present, PTH (1-34) has been widely used in clinical, mainly used in the treatment of osteoporosis [8,9].Natural PTH is composed of84amino acids (PTH (1-84)), and the I type PTH receptor (PTHR1) binding, N terminal34amino acid residues (PTH (1-34)) with PTH (1-84) the same PTHR1binding and activation function. Research shows that PTH regulate [10] links bone cell generation, transformation, proliferation or apoptosis, is reached via indirect effects on osteoblast on osteoclast function, its effect is a bone cell surface receptor type I PTH receptor (PTHR1)[11], leading to activation of multiple signal pathways of PTHR1and its downstream and regulate the bone.Therefore, whether the PTH (1-34) is applied to the bone metastatic cancer patients, using PTH (1-34) to promote bone formation and remodeling of bone structure function, delay the pathological fracture, and prolong the life time, become the preliminary idea in our experiments, and the realization of this idea, establish a animal model is the foundation we test.The preparation of animal model is the basis of bone metastasis, bone metastasis animal model cancer, establish a common established/grown in immunodeficient animal in vivo animal tumor cell lines. The mouse is recognized as the best choice of animal drug test, the mice models of bone metastasis from mature, its production method is usually divided into three kinds: the first kind produced by blood disseminated tumor bone metastasis model1, intravenous injection method: intravenous injection method and the clinical bone metastases arising mechanism does not match, seldom used. But recently, Teresa (12) reported, the MDA-MB-231cell line in nude mice after continuous subculture, selected strains of different cell clones, to these cell clones transfected Luciferase Report Gene, was found by in vivo imaging technology, B02clone with other cell cloning is not same, the cell line is the rapid formation of soluble ability of bone type bone metastasis. Results show that B02mice by tail vein injection,10days can form obvious bone metastasis.2, heart injection method:model and cancer patients with bone metastases in growth or morphology are very similar to produce left ventricular injection of bone metastasis, is currently one of the most common methods. The model not only has many advantages, but also conforms to the Paget’s "seed and soil theory":tumor cells as a "seed", the bone marrow cavity microenvironment as "soil". In support of the theory of evidence are:tumor cells via the left ventricle injection, selective growth and the formation of bone metastases in the bone microenvironment cells; inhibit osteolytic reaction can inhibit the metastasis of tumor growth. The preparation method of the model is the multiple metastatic brain, lung, adrenal cortex, can also cause some mice bone metastasis is not yet due to metastasis of other lethal. Second kinds of methods, local intraosseous injection preparation of bone metastasis model, this method will tip directly stab Tougu cortex, the backbone of transplanted tumor cells injected into the tibia or femur. With the tumorigenic rate is high, simple production method, and not subject to organ metastasis effect. Defect is caused by the injury of cortical bone and medullary cavity, and the production of model as the backbone of the transfer, common and clinically different epiphyseal transfer. Intraosseous injection method is often used to make into bone or bone/osteolytic bone metastasis model of mixed (13). The model provides a valuable mechanism and therapeutic effect of bone metastasis. Third kinds of methods, tumor orthotopic transplantation model, orthotopic transplantation refers to the cancer cells or tissue block cultivation in the mouse mammary fat pad of the chest wall or prostate tissue and bone metastasis. Retain the whole process from the primary tumor to metastasis to bone formation model with the method of bone metastasis, but the replication method also shortcomings, compared with the introduction of blood spread, intraosseous injection method, bone metastasis rate is low, the transfer and associated with other organ. So if the tumor, the tumor is easy to observe, not susceptible to organ transfer considerations, then second methods for preparation of animal model for application.In the background, select a mouse model of lung cancer with bone metastasis, and based on this model of intermittent subcutaneous injection of small dose of PTH (1-34), by observing the mouse body X-ray, micro CT, bone histopathology, serum ALP index, preliminary observation of PTH (1-34) effect on tumor growth of lung cancer bone metastasis of small rats.ObjectiveEstablishment of mouse model of lung cancer with bone metastasis;Preliminary observation of parathyroid hormone PTH (1-34) tumor of bone metastasis of lung cancer.Method304～6week old BALB/c female mice, method of suspension intraosseous injection with A549human lung cancer cells produce proximal tibia bone metastasis of lung cancer model,7days after the X-ray determination model was established, and randomly divided into three groups, one group was injected with parathyroid hormone PTH (1-34) as the observation group, a group of injection volume of solvent as the blank control group, a group of injection of cyclophosphamide as the positive control group, body weight was measured every2days one time and observe the right hind limb tumor growth, the mice were killed after28days, all mice underwent X-ray and CT examinations were observed tumor shape, size, CT detection of tibial bone density and tumor size, HE staining and immunohistochemistry histochemical method was used to observe the tumor morphology and properties, to detect the concentration of serum alkaline phosphatase to analyze osteogenesis. All data are presented as mean±standard deviation (x±SD) said, were analyzed using SPSS13.0statistical software, ALP, tumor size and CT data analysis to do the whole test with single factor variance, two two by LSD-t test, P<0.05was considered to be statistically significant differences.Result1, the tumor bearing mice models:this group30mice, after7days of X-ray examination, all the tumor. 2, three groups of mice weight: on the35day after modeling observation period, compared with the control group, the observation group mice weight slightly decreased at day14after using mode selection, after increasing with time gradually increased, in21days after modeling with control group and cyclophosphamide group, weight a slight decrease in the14day after modeling, body weight growth is slow, after35days than the control group and the experimental group.(Figure2)3, The activity level of ALP in serum:3, compared three groups of mice serum between baseline, ALP levels were significantly higher in group PTH (1-34) group, ALP higher than that of control group, there was significant difference between the two groups (p<0.05), PTH (1-34) and the control group increased significantly than cyclophosphamide group, there were significant differences between the groups (p<0.05).(Figure3)4, the X-ray manifestations of three groups:there were bone destruction, the observation group and the control group, bone destruction, showed osteolytic destruction, proximal tibia, obviously, the control group of1mice left tibial fracture, bone destruction and cyclophosphamide group significantly lighter, and6mouse cortical bone integrity, tumor less effect than before two groups.(Figure4)5, micro CT manifestations:observation group and control group were in moderate and severe tibial plateau bone destruction, with bony, soluble apparent destruction of cortical, medullary cavity filled with cancer, most of the trabecular bone. And the bone metastasis of lung cancer clinical features consistent with [14], cyclophosphamide group bone destruction and mild to moderate, cortical bone is still intact, growth was better than the observation group and the control group (Figure5) inhibition of bone tumor. Using CT software to detect bone tumor volume (Figure5), PTH (1-34) difference between bone tumor volume and control group no significant (p>0.05), while the two group of bone tumor volume was significantly higher than cyclophosphamide group, significant difference between groups (P<0.05), suggesting that PTH (1-34) did not inhibit metastasis tumor growth, but also did not promote tumor volume growth trend of bone metastasis. Detection of proximal tibia bone density tumor foci (Figure7), from Mean/Density of two BV and BV/TV data suggest that the PTH (1-34) bone mineral density was bigger than that of the control group, there was significant difference between groups (p<0.05, two), bone mineral density was decreased significantly in cyclophosphamide group, significant difference between groups (p<0.05).Conclusion1, Successful establishment of the tibia in BALB/c mice proximal metastasis model of human lung cancer A549cells,1, tibial plateau cancer cell suspension injection of tumor formation time, uniform size, high tumor formation rate (of the100%), is an ideal animal model of tumor occurrence, development, metastasis mechanism.2, ALP is the promotion of biochemical markers of bone formation, X-ray scanning, micro CT scanning and bone tissue biopsy is a good index in detection of bone metastasis:①The alkaline phosphatase (ALP) is a sensitive indicator of [15] evaluation of bone formation, three groups of mice after modeling, the metastatic tumor cells break down bone will stimulate the proliferation of osteoblasts on repair causes the increase of ALP, and the observation group with small dose of PTH (1-34) to further stimulate the osteoblast, further promote bone formation.②The X-ray can observe the whole bone structure in mice, can be in vivo x-ray scanning under anesthesia, benefit at different time points after modeling (1weeks and5weeks) to observe the tumor model photos, for early to observe the mold is formed, later can clearly determine growth and destroy the image of lower limb whole tumor.③The micro CT scanning clearly show the shape, bone tumor destruction of bone, trabecular bone and marrow cavity infiltration of3D reconstruction, displaying the tumor size, shape, range; using CT software measurement of tumor length, short diameter, through the formula v=length x short diameter2measuring tumor size, can analysis difference and tumor size were between of BV and BV/TV; analysis of Mean/Density data, BV and BV/TV is indicative of bone mass and bone fraction index, the comparison between two two groups, the difference between the two groups, the differences in the representation of bone.④The destruction of bone, tumor cells in the pathological, immunohistochemical resolution tumor nature, distinguish the original tumor or tumor.3, combining the above theory and the results of data, we conclude that, intermittent small dose injection of parathyroid induced PTH (1-34) does not promote the growth of mouse lung cancer bone metastases, but it can increase the surrounding bone metastases. |
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