Basic Medical Science Research Institute of China’s Academy of Medical Science
Li Mei(1) Zhang Shifu(2) Wu Hong
DongdanParkInstructionCenter of Beijing Zhineng Qigong Branch
Liu Jiahong(3) Li Fengchun
Use Flow Cytometer to analyze the synthesis of DNA and the cell cycle in the cells of human liver cancer (BEL-7402). The result of the experiment proves that cell growth in the experiment group is inhibited. The cells are checked and piled up in the pre-synthesis phase (Stage G1), as hinders the cells from entering the DNA synthesis stage (Stage S), thus disturbing the duplication of DNA.
Zhineng Qigong, flow cell luminosity method (FCM), DNA synthesis, cell cycle
We have proved in experiment that Zhineng Qigong can inhibit the growth of animals’ transplanted sarcomas and the multiplication of cancer cells cultured outside the human body. Yet the mechanism of the inhibition still needs exploration. In the experiment reported in this article, flow cell luminosity method (FCM) is adopted in the research and the analysis of Zhineng Qigong’s effect on DNA synthesis and on the cell cycle in the liver cancer cell in order to make a preliminary exploration into Zhineng qigong’s function on cancer inhibition.
Material and Method
Culture of Cells:
The cancer cells used in this experiment are the cell line (BEL-7402) of liver cancer in the human body established by Chen Ruiming, et al., in Shanghai Cell Biological Institute. They are epithelium-like malignant cells. We culture the cells in the culture medium of RPMI 1640 that contains 15 percent of calf blood serum. Experiment group and control groups are established which are cultured in two separate incubators. The experiment group receives qi transmitted by qigong masters, 3 min to 5min for each transmission. The control group is not treated by any special process. The experiment is done repeatedly for three times.
Fluorescent Coloring of DNA and FCM Measurement
Make cancer cell samples according to the special technical requirement of this measurement. After RNA enzyme digestion and with ProPidium Iodide (PI) fluorescent coloring, we measure the PI fluorescence intensity of individual cell at the speed of 1000 cells per second, with each sample being measured for at least 10,000 cells, using FACS 420 Fluorescence Stimulation Cells Divided Sorter（product of Becton-Dicknson Company）. The laser illuminator includes 2 watt argon ion laser, with the wave length at 488 nm. The PI fluorescence passes through 585nm interrupted filter. It is measured with photomultiplier tube and is analyzed with polygram pulse analyser. Scatter diagram and histogram of the DNA of the cells are shown. Data obtained are input into computer. Carry out curve fitting analysis according to FACS 420 software multi-normal fitting program and compute the DNA content so as to obtain the percentage of the cell cycle (G1, S1, G2+M) and the variation coefficient (cv%) of G1.
Statistics obtained from FCM measurement shows that Cancer cells in the control group are in a swiftly multiplying state. The cell numbers in the DNA synthesis period (Stage S) are higher. They are respectively 61%, 64% and 39% in the three experiments. The cell cycle in the experiment group is 35%, 40% and 27%. Yet, the number of cells in the experiment group in pre-synthesis phase (Stage G1) is 2 to 3 times higher than that of the control group. It is 35%, 40% and 48% in the experiment group, but only 10%, 19 % and 18% in the control group. This indicates that the growth of cancer cells in the experiment group is inhibited under the effect of external qi, therefore most cells are checked in their growth and piled up in Stage G1, as hinders the cells from smoothly going into Stage S, which consequently interferes the synthesis of DNA.
We have also studied the influence of external qi on the growth curve of BEL-7402. The result shows that cell multiplication in the experiment group is evidently inhibited. The experiment is done repeatedly twice with similar result that agrees with the result of FCM measurement.
We know that cell cycle is divided into four stages: 1) From the time when mitosis is finished to the time before DNA is duplicated, which is called Stage G1; b) Stage S when DNA is being duplicated and the content of DNA is twice that in Stage G1; c) From when the duplication of DNA is finished to the beginning of mitosis, which is called Stage G2. 4) From the beginning of cell division to the end of it, as is Stage M. When cell cycle is being analyzed, the biochemical method is adopted in measuring the average content of DNA in the cell mass, the method of autograph with the incorporation of 3H-thymidian is used to measure the parameter of cell cycle, or static cytophotometer is used to measure the content of DNA in a large number of cells and a histogram of the distribution of the content of DNA is drawn in order to analyze the cell cycle. These methods are not only time-consuming and reqire a lot of effort but usually can’t reach the accuracy degree required in statistics.
FCM is a comparatively new modern international technique that can quickly and accurately measure the content of DNA in every cell and can compute the transmigration change of cellgeneration cycle. The result of our experiment proves that the external qi causes the aggregation of liver cells BEL-7402 in Stage G1 and hinders the cells from smoothly going into Stages S, the DNA synthesis stage when the genetic substance is duplicated. Because of the interference of the external qi, the synthesis of DNA is affected and blocked. This is one of the mechanism that external qi inhibits tumor.
Although this experiment is preliminary, it provides a certain theoretical basis for Zhineng Qigong’s the mechanism inhibiting tumor.
Li Mei: Researcher
Zhang Shifu: Assistant Researcher
Liu Jiahong: Professorr