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6�"{$4%0d%�"�*@ �*�"�*7�"�V �$�$V d%���������������������������������������������������������������������*�	�:	Introduction
 Colon cancer has become the most frequently diagnosed malignancy and the fifth leading cause of tumor-related deaths in China with mortality rates ranging from 7.35/10 to 8.32/10 in men and 6.34/10 in women [1-3]. However, establishing a simple, suitable animal model for studying biological behavior and heterogeneity of colon cancer cells, the mechanism of tumor metastasis should be encouraged. After decades of research, different experimental modeling methods have been discovered to have various advantages and disadvantages,�which fall according to requirements of the particular experiments and chosen models, intrasplenic-planted models can generate isolated liver tumor model [4-7].
Xenotransplantation may be established in a diffuse liver metastasis model; (ectopic) intraspleen injection method can be a good imitation of hemorrhagic transfer[8-10]. In the selection for tumor source, the experiments confirmed by subcutaneous transplantation tumor cell lines that it is more stable and invasive. In addition, different animal models and the choice of cell lines is also vital, not all tumor cells and compatibility can successfully establish an animal model[11-12].This research adopts HT29 cell lines and tumor cell medium at the injection site, and surgical methods were analyzed, and subcutaneous transplantation tumor models were screened for optimal conditions. However, stable tumor has established the subcutaneous transplantation tumor of colon cancer, as well as repeated model provides the basis for the further modeling.
1. Materials and methods
1.1. Materials
1.1.1. Reagents: 
Dimethyl thiazolyl diphenyl tetrazolium (determined by MTT); Dimethyl sulfoxide salt DMSO (Sigma companies in the United States); RPMI1640 (Gibco, USA); Fetal bovine serum (hangzhou sijiqing company).
1.1.2. Material: 
Carbon dioxide incubator (NAPCO companies in the United States); Super clean workbench; Centrifuge; ELISA enzyme standard instrument (Bio-Rad company); Inverted microscope (Japanese company); Cell culture plate (Millipore company); Sterile filter (Costar companies in the United States); Sterile surgical instruments.
1.1.3. Cell lines: 
   We purchased colon cancer HT29 cell lines from the Chinese academy of sciences, Shanghai Institute of Cell.
1.1.4. Experimental animals: 
6 ~ 8 weeks of BALB/C mice, male, 18 ~ 22 g weight provided by Animal Experiment Center of Xinjiang Medical University (experimental animal product license: SCXK2003-0001). All animal research has passed the first affiliated hospital of Xinjiang Medical University animal experiment center ethical review committee for review.
1.2. Methods
1.2.1. Cell culture:
    Thaw HT29 colon cancer cell lines were washed twice with normal saline, in medium containing 10% newborn bovine serum (56 degrees Celsius inactivated for 30min) and RPMI1640 culture.  At 37 degrees Celsius and 0.05 CO2 culture incubators the vaccine cultivation in the liquid bottle, for adherent cell growth, remained cells grow to pave with culture bottle bottom more than 80% cell count, trypsin digestion. Adjust the concentration for every 0.2 mL medium, contains 2 x 107 cells (1�106 / mL), to observe the cell vitality (trypan blue dyeing rate of living cells) > 95 percentage, as set aside.
1.2.2. Subcutaneous transplantation tumor model of colon cancer screening:
Colon cancer HT29 cell line cell suspension was washed twice with physiological saline with PBS and serum-free culture medium heavy suspension. Cells 6 ~ 8 weeks of BALB/C mice were randomly divided into 4 groups laterally in axilla and dorsal subcutaneous inoculation. After heavy suspension, cells in subcutaneous tumor growth of about 1.5 cm3 were executed in mice skin disinfection with complete stripping subcutaneous tumor. Immediately immersed with 100 U/ml of normal saline penicillin and streptomycin of each group, eliminate the surrounding connective tissue and tumor growth organization. Cut up into 1.5 mm diameter fragments, again as a transplant source material using the enzymatic hydrolysis, homogenate methods for three groups of BALB/C mice vaccinated subcutaneously, Finally, subcutaneous tumor used as a source of tumor third-generation subcutaneous transplantation tumor model is shown in figure 1.
1.2.3. Anatomy and histopathologic observation
Subcutaneous tumor burdened in mice was checked in detailed anatomy and materials. It was discovered that the tumor had spread to the liver and other organs as well. Subcutaneous transplantation tumor was removed with 10% neutral formaldehyde fixed, dehydration, transparent, paraffin embedding and sectioning. HE staining and sealing piece were observed under light and camera.
1.2.4. The Immunohistochemical detection
     Slices were dried with xylene and dewaxing grading and were washed using distilled water for 10 min. With 3% H2O2 mixture (80% methanol), elimination of endogenous hydrogen peroxide enzyme activity was done for 10 min at room temperature. Immerse biopsy was taken after heating in microwave (> 94 degrees Celsius) of sodium citrate 0.01 mol/L (pH 6.0) buffer. Natural cooling at room temperature and then clean three times each for 5 min. With Super-Block, conducting liquid at room temperature closed with nonspecific targets for 1 hour to join the dilution ratio of first antibody, appropriate incubation for a night with the wet box 4. PBS cleaning three times, EnViSion System (HRP, mouse/rabbit) reaction resistance 37 degrees Celsius and 20 min incubation. PBS wash 5 times for DAB/H2O2 reaction configuration liquid controls dyeing degree under a microscope for 10 min, PBS resin 10 min, tap water rinse fully, hematoxylin counter stain 2 min, conventional dehydration, transparent, neutral resin sealing piece, in a microscope, detecting the expression of CK19 and P53.
1.2.5. Detection of flow cytometry
     Adjust the cell concentration to 1x106 / mL, with 1 mL cold PBS centrifugal washing, 800 r/min 5 min until fluid seeps out. After 100 �L icy PBS after heavy suspension, add 75% ethanol to cell suspension 200 �L (- 20 degrees Celsius pre cooling) fixed cell for 1 hour. Join with PI (final concentration 50 �g/ml) and no DNA enzyme pollution of RNA enzymes (final concentration of 50 �g/mL) 1 mL of PBS solution, 37 degrees Celsius, 30 min - 1 h, in dyeing grinder. Compute with the cell cycle ModFit software sampling for cellular DNA content and  cell cycle analyze table (Fujiwara et al., 2012).
1.2.6. Statistical methods
      The generation of subcutaneous tumor model was selected by use of factorial analytic method; the second generation of subcutaneous tumor model of group repeated selected for the measurement of data analysis. All statistical data and analysis were conducted by using SPSS16.0 statistical analysis and calculation.
2. Results
2.1. The cell culture results
     Human colon cancer cell lines HT29 growth, which contains 10% fetal bovine serum, and RPMI1640 culture medium, including green streptomycin, is put in 37 degrees Celsius containing 5% CO2 culture incubator, and the cells grows well in a small group, as shown in figure 1 and figure 2.
     The injection site is divided into the following categories; axiliary and dorsal subcutaneous. Serum-free cell culture medium is divided in PBS and divided into 4 groups, each group having 5 mice only. After 18 consecutive days, the following data was concluded, as shown in Table 1:
     After analysis by SPSS, the difference between tumor cells and the cell media time was statistically significant (F = 82.526, P = 82.526) with PBS than with serum free medium into tumor extended by 2.8%. Different injection into the tumor time difference is not significant (F = 3.789, P > 3.789), the back of the neck than axiliary hypodermic injection into the tumor on time but there was no difference in the time of change. Interaction has no statistical significance (F = 2.13, P = 2.13) or at the injection site effects on tumor time has nothing to do with the cell medium, or use the PBS or medium in auxiliary or on the back of the neck after injection into the tumor but there was no difference in the time of change (Rashidi et al., 2005).
2.2. The model operation methods of selection subcutaneous transplantation tumor 
According to different batches, the method is divided into three groups that include insert blocks, homogenate and enzymatic hydrolysis, with each group having 5 mice only (Hackl et al., 2013). Using these three methods in 9 days, 12 days, 16 days and 20 days subcutaneous tumor, the size of the tumors had been statistical analyzed and the results are shown in Table 2:


The differences between tumors size are not at the same time phase (F = 1.600, P < 0.01), size of the tumors had no statistical difference, namely (F = 0.772, P > 0.772), no interaction between different methods and time of tumor (F = 1.035, P > 1.035).
2.3. organization in general and pathological biopsy examination
Biopsy is poorly differentiated with no obvious glandular structure. Cancer has different organization forms, more show elliptic, abundant cytoplasm, nuclear deformity, hyper chromatic, fission like and diffuse. Invasive growth can be seen as a connective tissue infiltration between cancer cells, as shown in figure 3 and figure 4.
2.4. The immunohistochemical method to detect
Microscope subcutaneous transplantation tumor, according to the shaded part of positive cells and detection of the expression were of CK19 and P53 types. CK19 and P53 protein positive staining in the cell nucleus were in brown (Rashidi et al., 2005). The total number of positive cells under 25% for weakly positive, 25% ~ 49% for moderate positive, greater than or equal to 50% were strong positive. As shown in figure 5 and figure 6.
2.5. Flow cytometry detection
      Based on HT29 cell lines, subcutaneous transplantation tumor DNA cycle times and analysis, the results are heteroploid, as shown in table 3.
3. Discussion
 Our research has chosen those individuals with HT29 colon cancer cell lines, because these cells are homologous of colon cancer in the human body, and it is a primary tumor stimulation process in the body. In addition, the early experimental study has confirmed that colon cancer HT29 cell lines have stronger invasive and proliferation ability to survive in the cell culture and living microenvironment with short incubation time, and none can proliferate. BALB/C mice have stronger survival ability in small, easy breeding, and the price is low. They have also been widely used in the animal experiments[13-15]
According to Boni et al., (2005), subcutaneous transplantation tumor model is successfully influenced by many factors, in addition to tumor cells and the selection of experimental animals, including injection quantity, injection site of tumor cell, media, etc. Acquired factors include the following categories:
 1) the number of tumor cells inoculation are not too high or low in volume, transplanted cells success rate, less quantity and spontaneous transfer rate will be decreased.  Too much cell quantity will not only cause out of control tumor growth, but it can also cause cell suspension fluid�leakage after withdrawal needles. It will make subcutaneous tumor form in different shapes. The experiments were made to select each cell inoculation quantity and should be 1 x 106 / mL with 0.2 mL of cell suspension.
 2)  choice of vaccination site for subcutaneous tumor:  this experiment was compared to  the position of the inoculation, according to the results of the armpit and subcutaneously back of the neck inoculated initially in tumor time has no obvious difference, but in the process of growth of tumors. We found that the underarm pushed backward as the tumor grew because one of the auxiliary vessels has rich blood supply for tumor growth. Secondly, arms with flabby skin can make larger tumor nodules. One needs to pay attention to the operation of the vaccination site to precisely control under the skin. In our experiment, the experience of intradermal adhesions with skin, muscles and into the abdomen was easy to make tumor padded grind rotten has burst. 
3) cell medium choice: this experiment chose different cell group contrast medium, the results after using PBS suspension of subcutaneous tumor is serum free medium suspension after subcutaneous tumor growth is slow. PBS was used as cell medium, because of the lack of vitamins and amino acids reflect chances of tumor characteristics later, makes the advantageous study of liver metastatic tumor angiogenesis and proliferation .A cell with culture medium will provide sufficient nutrition for tumor cells, makes the normal characteristics of tumor cells for early appearance and very quickly large cell concentration in  the tumor growth.
For the method of using continuous subcutaneous transplantation tumor to extend the selection, this study compared the insert block method with homogenate method and enzyme methods. After inoculated with three methods of tumor growth, results shows that the tumors had difference in sizes between different times (F = 1.600, P < 0.01), three methods from the size of the tumors had no statistical difference between (F = 0.772, P > 0.772). Moreover, we think that the keystone features of the successful models�are the three methods and the surgical techniques have certain contact.
  (1) the insert block method refers to the subcutaneous tumor chopped into pieces, then use insert block needle or hypodermic needles to block the tumor in mice. Our experience in the process of operation is as follows:
 we will choose uniform tumor tissue blocks all sequential placed in 3-4 vaccination needle, one-time to preoperative readiness of mice vaccinated, so that we can avoid time-consuming and in turn of vaccination can save the time of tumor in vitro.
 in addition, we discuss the improvement of this method, first into 0.5 cm incision in needle into place so that you can avoid needle tugging on the skin at the time of the needle. 
(2) the main problem is speed. The method in tumors in vitro to vaccination takes 1 hour to complete, as far as possible to avoid the loss of the activity of tumor cells. Diction, homogenate method refers to the tumor cells into homogenate in grinding, after subcutaneous injecting mixed suspension into mice
Operation process in our experience is: (1) need to pay attention to the quality of slurry, we chose to use ground rod joint it is not tight in glass homogenate, clockwise grinding , control within the 3 times, tissue suspension after grinding with 200 mesh sieve. (2) Precisely control the needles as inserted under the skin, prevent the forming mixed tumor. 
4. Conclusions
This research is the subcutaneous transplantation tumor model in the process of the establishment of the cell medium, the injection site and operation method of screening. The analysis of the process is a success and builds convenient operation with colon cancer subcutaneous transplantation tumor model of repeatability to serve as a source of tumor. For further research on other modeling approaches, it provides ideal animal model of the foundation, also for the subsequent test provide a good animal model and platform.


Conflict of interests
Authors declare that they have no conflicts of interest.
Acknowledgments
This work was supported by grants from the General surgery clinical national key subject construction projects and Natural Science Foundation of  Xinjiang  science Department (No. 2014211C036).
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