| BACKGROUNDRecently, the treatment of tumor differently by traditional Chinese medicine(TCM),that can lengthen the life-span, improve the life quality,relax the clinic symptoms,weaken toxin and enhance effect with the program of radiotherapy and chemotherapy for the patients with tumor,has been confirmed by plenty of clinical research results.For the synthetic advantage of clinical effect, a synthetic evaluating system on tumor effect,that takes patients as the foremost,allows living with tumor,focuses on lengthening patient's life-span, improves life quality, alleviates clinical symptom in practice of preventing and treating tumor,has been proposed by tumor researchers.This new medical suggestion, that makes TCM evaluating system of clinical effect with main subjective index objectify, is a challenge to the evaluating system of measuring tumor curative effect with only standard of the change of tumor body treated mainly by operation, radiotherapy and chemotherapy. The excellent clinical effect benefits mass patients with tumor and arouses interest of tumor researchers at home and abroad. Therefore,the study of trying to search for new anti-tumor medicine from natural herbs has become a new popularity. The latest scientific technology, the most precision experimental instruments are promptly applied to the study of TCM. No less herbs with anti-tumor function have been screened out,many active elements with anti-tumor function have been extracted from herbs,the anti-tumor mechanism of TCM have been identified one by one.However, the results of studying anti-tumor Chinese medicine by modern means of science and technology can neither promote both Chinese and west medical theory, nor direct both clinical practices,nor create a new medical theory besides proving that Chinese medicine has synthetic effect on anti-carcinoma and restrain-carcinoma from multistage, multilink, multipoint.The reasons include:1.TCM focuses on individual dealing dynamic program of giving treatment in accordance with local conditions,seasonal conditions and patient's individuality, but west medicine mainly lays stress on static common treating program.2. West medicine seeks for pharmacological function of active facts,but it is compound and mixed elements in TCM that have effect on tumor body.3. West medicine takes illness seriously, aims at killing carcinoma cells, but TCM takes patients as the foremost,focuses on keeping the primordial QI in body, allows long-term existence with tumor.It proves that any study without directing of TCM theory, no matter how it is so advanced and scientific, will have to result in separating itself from clinical practice, deviating from original theory, eventually making subject degenerate. Undoubtedly,the TCM mechanism of tumor happening must be firstly defined before anti-tumor mechanism of TCM is studied. OBJECTIVE This study is to determine the kidney-deficiency mechanism from telomere on tumor happening through observing the effect of serum containing YOUGUIYIN compound on morphology, telomere dynamics, the activities of telomerase and tankyrase,the expression of mtP53 in human lung adenocarcinoma A549 cells.1.To express the kidney-deficiency mechanism of tumor happening by scientific data.2.To build up the anti-tumor theory of TCM possessing scientific property, scientifically and effectively directs clinical practice and experimental study of anti-tumor.3. To enrich and develop the TCM theory by the level of cell biology and molecular biology.METHODS AND RESULTS1.The kidney-deficiency mechanism from telomere on tumor happening is studied by observing the effect of YOUGUIYIN containing serum on morphology and function, telomere dynamics,the activities of telomerase and tankyrase and the expression of mtP53 in human lung adenocarnocima A549 cells.2.The results that the changes of morphology and passage on human lung adenocarcinoma A549 cells are observed by inverted microscope, and the senescent cells are detected by the experiments about the uptake rate of human lung adenocarcinoma A549 cells to [3H]-TdR and the transfection efficiency of human lung adenocarcinoma A549 cells on X-Gal show:after dealing with different doses of YOUGUIYIN containing serum, the uptake rates of human lung adenocarcinoma A549 cells to[3H]-TdR become gradually decreased,after 7 passages,the uptake rates of human lung adenocarcinoma A549 cells to [3H]-TdR in 30% YOUGUIYIN containing serum group came down to(6.78±0.92)%,and to(37.49±2.68)%,(34.33±2.59)% respectively in 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group, but the uptake rates of human lung adenocarcinoma A549 cells to [3H]-TdR in control group was(84.84±4.86)%,the difference among groups was significant (P<0.01),the transfection efficiencies of human lung adenocarcinoma A549 cells on X-Gal become gradually increased in each group,after 7 passages,the transfection efficiencies on X-Gal in 30% YOUGUIYIN containing serum group came up to(87.04±4.47)%,and to (34.55±2.75) %,(37.85±1.96)% respectively in 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group,but the transfection efficiencies of human lung adenocarcinoma A549 cells on X-Gal in control group was(6.20±0.89)%,the difference among groups was significant (P<0.01),the duration of passage became gradually lengthened, the passage times became gradually reduced,and the senescence and apoptosis cells become gradually visible in each group, the passages end, the senescence and apoptosis cells appear in the 7th passage for 30% YOUGUIYIN containing serum group, and in the 17th and the 15th passage for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively,in the inverted microscope, appear the senescent and apoptotic cells,few of adherent cells growing, a large number of cell with gradually shrinking cell membrane due to dehydration, rare pseudopodia around the cells, the cell shape changing into flat and swell,that more particles can be seen within the cytoplasm, that the cell membrane was integrity, that the cell plasma became dense, that the dense organelles were visible, the more common cells with color fade chromatin, that the chromatin in nuclear change into dense, forming clumps with different shapes and sizes,coming to the boundary of the nuclear membrane,or the crescent bodies were formed, by Hoechst33342 staining,showing reduced nuclear due to dehydration, and irregular nuclei fragmentation phenomenon, nucleus cracking, multiple cytoplasm bud, that with the cytoplasmic bud off the sudden, the apoptotic body cells were formed.3. The results that the telomere length in each passage cells are measured by Q-FISH show:after dealing with different doses of YOUGUIYIN containing serum, the telomere length becomes gradually shortened, the loses of telomere length in cell passing become gradually increased in each group,the loses of average telomere length in each passing for 30% YOUGUIYIN containing serum group reach 1.29±0.12kb, and 0.53±0.11kb,0.57±0.03kb for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively, compared with each group, showed statistically significant difference (P<0.01).4.The results that the telomerase activities are measured by PCR-ELISA show:after dealing with different doses of YOUGUIYIN containing serum, the telomerase activity in each group becomes gradually reduced, the telomerase activity for 30% YOUGUIYIN containing serum group is 36.44±2.49,and 64.75±2.63, 62.50±2.64 for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively in the 7th passage cells,the difference among groups was significant (P<0.01). 5.The results that the tankyrase activities are measured by Western Blot show:after dealing with different doses of YOUGUIYIN containing serum, the tankyrase activity in each group becomes gradually reduced, the tankyrase label at the spot of 142ku molecular weight,standard protein, for 30% YOUGUIYIN containing serum group, fades markedly more than other two groups in the 7th passage cells.6. The results that the mtP53 protein is measured by S-P immunohistochemistry show:the number and the colors of the positive A549 human lung adenocarcinoma cell with S-P immunohistochemical staining were observed in the ordinary optical microscope with magnification of 400 times,light through the spherical cell on the slide,the cytoplasm, organelle, the nucleus is clearly visible, mutant P53 gene protein(mtP53) mainly clustered around the nucleus,the colors of brown and red, brown and yellow, and yellow appear on the nucleus and around the nucleus in positive staining of A549 human lung adenocarcinoma cells with the number of the mtP53 protein expression, it can be seen that the expression of the mtP53 gene protein in the control group cell nucleus was the strongest,with different concentrations YOUGUIYIN containing serum treatment, the expression of mtP53 gene protein in A549 human lung adenocarcinoma cell nucleus gradually decreased,specially the expression of mtP53 gene protein in 30% YOUGUIYIN containing serum group was decreased most significantly. According to the observed number and colors of positive cells,the comprehensive evaluation results show that:in the control group,5 high power fields on each slide were observed, the number of positive cells was greater than 75%, score 4 points,the color intensity of staining is brown and red, score 3 points,according to rating standards,comprehensive evaluation was in the range of 9 to 12 points,indicating that mtP53 gene protein expression in the nucleus was strongly positive(+++).In 10% and 20% YOUGUIYIN containing serum group, five high power fields on each slide were observed, the number of positive cells was in 51% to 75%, and the score was 3 points,the color intensity of staining was brownish yellow, a score of 2 points, according to rating standards,integrated score was in the range of 5 to 8 points, indicating that mtP53 gene protein expression in the nucleus was moderately positive(++).In YOUGUIYIN 30% serum containing group, 5 high power fields on each slide were observed, the number of positive cells was in 26% to 50%, and a score of 2 points,stained brown and yellow or yellow color intensity, a score of 1 to 2 points,according to rating standards,integrated score range of 1 to 4, indicating that mtP53 gene protein expression in nucleus was weakly positive(+).After dealing with different doses of YOUGUIYIN containing serum, the expression of mtP53 protein in each group becomes gradually reduced, the synthetic score of mtP53 for 30% YOUGUIYIN containing serum group is 3.78±0.94,and 6.39±1.29,6.22±1.11 for 10% YOUGUIYIN containing serum group and 20% YOUGUIYIN containing serum group respectively in the 7th passage cells,but the score is 11.50±1.15,compared with each group, showed statistically significant difference (P<0.01).CONCLUSIONS1.The scientific implication for kidney dominating store-essence is: (1) keeping the energy of body steady, (2)keeping the space and the time of telomere in cell steady.2.Kidney-deficiency, dysfunction of the kidney in storing essence, exhaustion of body energy, is the rooted reason of tumor happening.3. Mutation of P53, being activated of telomerase system, unsteadying both space and time of telomere, is the telomere mechanism of tumor happening.4.Reinforcing the kidney can increase the losses of telomere length of carcinoma cells in each passage so as to keep the space of telomere steady, and reduce carcinoma cell passages so as to keep the time of telomere steady.5.Reinforcing the kidney can keep the space and the time of telomere steady so as to keep the energy of body and cells steady accordingly.6. Reinforcing the kidney can inhibit P53 from mutating, repress telomerase activity, prevent immortal and carcinoma cells from happening.7.Kidney-deficiency mechanism from telomere on tumor happening is that kidney-deficiency, dysfunction of the kidney in storing essence,exhaustion of body energy, unsteadying both space and time of telomere,mutation of P53,being activated of telomerase system, cell obtaining the ability to limitless proliferation. |
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