Experimental Study On The Effects Of Hydroxyapatite Nanoparticle In Human Renal Cell Carcinoma 786-0 Cells

Renal cell carcinoma, another name is renal carcinoma, is the most familiar renal parenchymatous malignant tumor, arrives at the 2nd in the tumors of urogenital apparatus (second only bladder cancer), occupies 2%-3% in all adult tumors and 90% in renal malignant tumors. Clear-cell carcinoma occupies 85% in all renal cell carcinomas. The incidence ratio of renal carcinoma increases on 3% in Europe and American, has no precise statistic in China at present but the crude mortality of renal tumors is 0.32/10 ten thousand people according to analyzing the structure of death reasons in China in the 1990s and the number is lower than the occident's. The incidence of renal carcinoma becomes to go up in recent years. The patient of RCC has no obvious symptom at earlier period. Over 50% patients have had metastasis or recurrence after operation visiting the doctor. The average survival time of metastatic RCC is 13 months, the survival rate of three years less than 5%. The systemic therapeutic method is extremely limited and the curative effect is not well in progressive RCC even though the RCC can be discovered early and cured promptly by modern diagnosed way and the survival rate of 5 years is higher than ago. The surgical is pivotal in the therapy of RCC, but the effect of chemotherapy is insensitive because the RCC produces multidrug resistance to cytotoxic drug and the RCC is not sensible to radiotherapy. That constructing one method of simple,safe and effective aiming at RCC becomes the choke point of preventing and curing RCC. We hope to explore a new way of curing RCC.Nanometer technique is pivot power among medical development at present, especially at the prevention and cure of tumor, which becomes the hotspot nowadays. The hydroxyapatite nanoparticle can inhibit the growth of cancer in recent researches, but the domestic research is few about the effect of hydroxyapatite nanoparticle to urinary system and is rare to RCC. We select 786-0 cell train of RCC as the research object at this experiment and discuss the action and mechanism of hydroxyapatite nanoparticle to RCC.I.Detecting the impact to growth and apoptosis about hydroxyapatite nanoparticle aiming at RCC in vitro.The experiment is divided into 4 test groups according to the density of hydroxyapatite nanoparticle (100,200,400 and 800μg/mL) and one control group (hydroxyapatite nanoparticle is absent). Combining with 786-0 cell for 24,48 and 72 hours, we detect cellular proliferation or inhibition by MTT, observe cellular morphological change by HE staining, observe ultrastructural change by transmission electron microscope, inspect cellular apoptosis at 24,48 and 72 hours with hydroxyapatite nanoparticle (200μg/mL) by FCM, detect cellular protein expression level of caspase-3,caspase-12 by immunohistochemistry SP method, detect the gene expression level byRT-PCR. The results show below:1 The results of MTT show that the growth of 786-0 cell train is inhibited when the cell is combined with hydroxyapatite nanoparticle for 24-72 hours which has density and time dependence. The inhibition ratio reaches to the tiptop when the cell is combined with hydroxyapatite nanoparticle (800μg/mL) for 72 hours. The growth inhibition ratios of each HAP group have significant difference with control group (P﹤0.01).2 The results of HE staining show that the cell turns round,crimple and small, the karyon is stained thickly, the chromatin becomes condensed or ruptured, the karyon gets pyknosis in ring,clumping or crescent, the karyon has fragmentation etc. The cell size of control group is always even, the cell membrane is integrated and the karyon is dyed lightly and evenly. The outline of karyotheca are obvious, the nucleolus is clear and has energetic caryocinesia.3 The control cells are discovered that by transmission electron microscope: the cell organ is normal, the cell surface has microvilli, the karyon is irregular and the nucleoli are obvious. There are many chondriosome,free ribosome,rough endoplasmic reticulum and few lipid droplet in cytoplasm. The test group cells: the karyoplasm is condensed,shrinkage,broke and forms apoptotic bodies. Hydroxyapatite nanoparticle appears inside and outside the cell. The necrosis cell has some changes: the membrane is broken, the content overflows, the karyotheca is broken, karyolysis, some karyons are crescent,ring or shivered, the endoplasmic reticulum is expanded and the vacuolation becomes more.4 The results of FCM show that apoptotic peak is present, the apoptosis ratio is 4.6%,8.3% and 10.7% respectively when hydroxyapatite nanoparticle (200μg/mL) is combined with 786-0 cell for 24,48 and 72 hours. So, the apoptosis ratio increases with acting time of HAP and has significant difference with the control group (P<0.05).5 The protein expression of caspase-3 in 786-0 cell is downregulated and the caspase-12 is upregulated when hydroxyapatite nanoparticle of various densities act for 24 hours, and the change is dependent with concentration. The protein expressions ( caspase-3,caspase-12) of test groups have significant difference (P﹤0.01)comparing with that of the control group.6 The agarose gel electrophoresis of RT-PCR product reveals that the gene expression of caspase-3 downregulate and caspase-12 upregulate along with the rising density of hydroxyapatite nanoparticle when 786-0 cells are combined with hydroxyapatite nanoparticle of various densities for 48 hours.The results of 3 pieces of PCR by photodensity scan reveal that the relative value of photodensity integral value of caspase-3/β-actin becomes downregulated with the upregulated density of hydroxyapatite nanoparticle ( P<0.01), and the value of caspase-12/β-actin becomes upregulated with the upregulated density of HAP( P<0.01).The experimental results show that hydroxyapatite nanoparticle may inhibit the growth of 786-0 cell of human renal cell carcinoma train and induce cell to apotosis in vitro. There are relations with upregulated protein expression and enhanced activity of caspase-12. At last, it can induce cell to apoptosis but the concrete mechanism need to be more studied.II Researching the biological behaviour of hydroxyapatite nanoparticle to 786-0 cell train of renal cell carcinoma1 Detecting the apoptosis of 786-0 cell by annexin V/FITC and PI double staining methodThe result shows that the apoptosis ratio of 786-0 cell acted for 24 hours has significant difference (P﹤0.01)between the test group (hydroxyapatite nanoparticle 200μg/mL) and the control group by LSCM, the apoptosis ratio is (4.88±0.15)% and (1.24±0.07)% respectively.2 Detecting migration capacity of 786-0 cell acted with hydroxyapatite nanoparticle by scarification test The result shows hydroxyapatite nanoparticle can restrain the migration of 786-0 cell.3 Inspecting the invasion capacity of 786-0 cell acted with hydroxyapatite nanoparticle by basal membrane invasion test (Transwell cave method) The result shows that hydroxyapatite nanoparticle can inhibit the invasive capacity of 786-0 cell in evidence.The experiment reveals that HAP can inhibit the invasion and metastasis of tumor cell (786-0 cell train) but the mechanism in detail need to be more researched.III.Detecting the growth change of 786-0 cell acted with hydroxyapatite nanoparticle by some tests in vivoConstruct a xenografted tumor model of mice and divide 20 Balb/c male mice of bearing tumor into 2 groups at random. Inoculate 1mm3 fibrin clot composed with 786-0 cell or 786-0 cell adding hydroxyapatite nanoparticle to below renal peplos. Sacrifice mice after 7 days and contrast the tumor cellular infiltrative depth. The result shows that the depth of 786-0 cell group exceeds that of the 786-0 cell adding hydroxyapatite nanoparticle group(P<0.01).Discover that tumor tissue invades into the renal cortex at various degrees by 40×microscope with HE staining and the depth of 786-0 cell adding hydroxyapatite nanoparticle group is less obviously than 786-0 cell group.Examine the expression of the calpain by immune fluorescence staining and the result shows the value of 786-0 cell adding hydroxyapatite nanoparticle is higher obviously than 786-0 tumor cell group(P<0.01).The experimental results show hydroxyapatite nanoparticle can inhibit the growth of 786-0 cell strain (human renal cell carcinoma) transplanted under renal peplos of mice. The capacity may have relations with upregulated expression of calpain in xenografted tumor and activated caspase-12. At last, the cancer cell appears apoptosis,degeneration and necrosis so that the xenografted tumor is inhibited. The invasive depth to normal tissue is less than the control group in evidence. So, the hydroxyapatite nanoparticle can inhibit efficiently the invasive capacity to round tissue, consequently, inhibit local infiltration to renal carcinoma tissue.