Anti-tumor Effects Of Hydroxyapatite Nanoparticles Against Chordoma Cell Lines In Vitro And Vivo

Objective: 1.Extract primary chordoma cells from surgical specimens of patients,gradually purify them,establish cell lines,and explore the growth rules of chordoma cell lines.2.Explore whether HANPs have anti-chordoma effects through in vivo and in vitro experiments,and the potential mechanism.3.Explore the biological safety of HANPs through in vivo experiments.Materials and Methods: 1.Establishment and identification of primary cell line of chordoma:(1)Specimens of patients with chordoma in the clinic were collected,by using collagenase/hyaluronidase digestion method(collagenase method)and tumor tissue single cell enzyme digestion method(single cell method),the chordoma cells were obtained;(2)The differential adhesion method was applied to gradually purifying the primary chordoma cells to complete the establishment of the cell line(named WCH);(3)HE staining,immunohistochemistry and flow cytometry were used to identify the extracted cells,the results were compared with mature cell lines MUG and U-CH1;(4)WCH tumor-bearing nude mice were established.Specimens were obtained for HE staining and immunohistochemistry,and then compared with patients’ specimens to evaluate its tumor-forming ability.2.Experiments of HANPs anti-tumor against WCH,MUG and U-CH1 cell lines in vitro:(1)The effects of HANPs on WCH,MUG and U-CH1 cells were explored via CCK8,Ed U experiment,flow cytometry of apoptosis and cycle experiment in vitro;(2)Transmission electron microscope observation the position of HANPs after entering the WCH,MUG and U-CH1 cells and the affected cell organelle.3.Experiments of HANPs anti-tumor against WCH,MUG and U-CH1 cells in vivo:(1)Animal models of chordoma cells were established by subcutaneously injecting WCH,MUG and U-CH1 cells into the right back of Balb/c nude mice,respectively;(2)Results: 1.Results of establishment of primary chordoma cell lines:(1)The single-cell method was significantly better than the collagenase method in the isolation and extraction of chordoma primary cells.The positive expression rates of Brachyury in the cells extracted by the two methods were 82.9% and 32.9%,respectively;(2)After 7 times of differential adhesion,the cells were clearly purified,under the microscope,the chordoma cells grew uniformly,and no fibroblasts were seen;(3)The flow cytometry showed that the positive expression rate of Brachyury in purified WCH cells was 99.34%,and the corresponding MUG and U-CH1 were 99.06% and 99.86%,respectively,which was significantly higher than the initial rate of 82.9% before purified.HE staining of the extracted cells showed a large number of vacuoles and abundant cytoplasm.Cellular immunohistochemistry of CK,vimentin and Brachyury had a strong expression while the EMA,S100 were slightly weaker,which were consistent with the results of MUG and U-CH1,and also consistent with the cell-derived histopathological results.(4)Chordoma model of Balb/c nude mice were successfully established,the pathological examination results of the specimens were exactly the same as its originate.The chordoma cell line has been successfully established,which has been passed down to the 18 th generation.2.The results of HANPs anti-tumor against WCH,MUG and U-CH1 cells in vitro:(1)CCK8 results indicated that the inhibition of HANPs on the viability of WCH,MUG and U-CH1 cells was roughly time-dependent,and the optimal concentration was about 480μg/m L.(2)Ed U results of WCH and MUG cells showed similar concentration-dependent proliferation,while U-CH1 cells showed concentration-dependent proliferation on the 3rd day,except for the 5th day.Except for the obvious proliferation in the concentration group(60μg/m L),the rest were basically the same as the blank control group;(3)The cycle results showed that the cell cycle ratio in WCH cells was lower in both the low concentration and high concentration groups(960μg/m L)than in the middle concentration group,and decreased with the increase of culture time;In MUG cells,the cell cycle ratio increased as the concentration of HANPs increased;In the U-CH1 group,as time increased,the cell cycle ratio of the high-concentration group increased significantly,while the ratio of the experimental groups with the intermediate concentrations were equivalent,but all were higher than the blank control group;(4)Apoptosis results showed that in WCH cells,the apoptotic rate of 1st day 3rd day in low-concentration group and high-concentration group were higher than the intermediate concentration group.However,the 480μg/m L concentration group hold the highest apoptotic rate on the 5th day.(5)Transmission electron microscopy showed that after entering the cell,the HANPs were distributed in the cytoplasm.The mitochondria were swollen or pyknotic to varying degrees.The continuity of some cell membranes was interrupted.3.(1)Balb/c nude mice of WCH,MUG and U-CH1 cell animal tumor models were successfully established;(2)Compared with the blank control group and μ-HA control group,the growth of the WCH tumor in the HANPs group was significantly inhibited.There is a difference between the blank control group and the μ-HA group(P<0.05);(3)In both MUG and U-CH1 tumor-bearing nude mice,the tumor volume was the largest in the blank control group at the same time point,followed by the μ-HA group,while the DDP group was comparable to the n-HA group,significantly smaller than the blank control group and the μ-HA control group(P<0.05);(4)HE staining showed that WCH,MUG and U-CH1 blank group having a deep nuclear staining and a large number of vacuole-like cells;immune group chemistry showed that the expression rate of ki-67 in the WCH blank group(15.20%)was higher than that in the μ-HA control group(9.34%)and the n-HA experimental group(9.13%);In MUG,except that the DDP group was negative.the blank group is comparable to the μ-HA control group,and both were higher than the DDP control group and the n-HA experimental group,and the latter two are similar;In U-CH1,the μ-HA control group was slightly higher than the blank group,Both were higher than the DDP control group and the n-HA experimental group;(6)There were no abnormal values of the blood routine and biochemical examination of nude mice in the WCH,MUG and U-CH1 groups(P>0.05).(7)No organic damage or inflammatory reaction were found in the important organs of heart,liver,spleen,lung and kidney in each group.Conclusion: 1.The primary chordoma cells were successfully extracted and purified,and a chordoma cell line was established,which has now passed to the 18 th generation,laying the foundation for the follow-up HANPs anti-chordoma research.2.In vitro and in vivo experiments showed that HANPs inhibited chordoma cell lines and chordoma to varying degrees,and the best in vitro concentration was 480μg/ml.The possible mechanism is that HANPs cause mitochondrial swelling and pyknosis after entering the cell.3.In vivo experiments showed that HANPs had good biological safety.It is expected to become a new material for preventing local recurrence of patients with chordoma tumors after surgery.