Observation with Transmission Electronic Microscope
China’s Academy of Medical Science
Basic Medical Science Institute of ChinaXieheMedicalScienceUniversity
Qiang Liping Zhang Jinbo
DongdanParkInstructionCenter of Beijing Zhineng Qigong Branch
Liu Jiahong Wang Ximing
This article is about the observation of Zhineng Qigong’s effect on the super micro structure of tumor cells. The result shows that Zhineng Qigong, functioning on cells Hela, is not only able to stimulate and bring about the death of the cell program (cellapoptosis), but seems to be also able to induce the necrosis of the cells at the same time. The two factors that lead to the death of the cells are combined and inhibit the limitless multiplying ability of tumor cells.
Zhineng Qigong, cell Hela, apoptosis
Qigong therapy that has been enjoying a history of thousands of years in China has also a rather profound foundation. It is now attracting more and more recognition throughout the world. There is a great variety of qigong, however. We have reported the influence of Zhineng Qigong on the formation ability and the multiplication ability of cell Hela’s clony in human cervix cancer (Gao Cuihua, et al., an article waiting to be published in Oriental Qigong). It has been proved that Zhineng qigong has an inhibiting and killing effect on tumor cells (Cao Xuetao, an article published in Nature, 12:190, 1989). The aim of this experiment aims at observing the influence of Zhineng Qigong on the super micro structure of tumor cells.
Material and Method
- Hela cell lines are kept in cryopreservation in liquid nitrogen in our laboratory. They are swiftly revived at 420C and are cultured in an incubation box at 370C with 5% CO2.
- The culture system of RPM11640 culture medium (bought from GIBCO Company) contains 10 percent of calf blood serum (bought from Biochemical Reagent Company of Blood Research Institute in China’s Academy of Medical Science), 100u/ml penicillin and 100ug/ml streptomycin (both bought from Shanghai No. 3 Pharmaceutical Factory).
- Generation Culture
Let the multiplication happen when the cells are at the increased logarithmic phase. Inoculate 2×104 cells in each culture bottle with a culture area of 25 cm2, and inoculate altogether 16 bottles. Divide them randomly into two groups, with 8 bottles of cells in each group. Let one be the control group and the other the qigong group. Change new culture medium into the bottles every two days.
- Function of qigong on the cells and the counting of their number.
From the day when the cells are inoculated, five Zhineng Qigong masters begin to organize qi field together in four successive days. They transmit qi with their mind once a day, and each period of qi transmission lasts for 15 min. Observe the growth of the cells in the two groups with light microscope. Collect the cell suspension respectively when the cells have been cultured for 24h, 48h and 72h since the first qi transmission and wait to treat them with the method recorded in 5. At the same time, take one bottle from each group everyday. Digest the cells that have become deposited and count the rest cells that have become adhered to cell walls.
- Collect cells and observe them with electronic microscope.
Collec the cell suspension of each group respectively when the cells have been cultured for 24h, 48h and 72h since the first qi transmission. Centrifugalize it for 5min at 1000rpm and deposition of clone can be seen at the bottom of the bottle. Wash the cells with equilibrium liquid PBS (PH7.4). Move them into eppendorf tube and centrifugalize them in the way mentioned above. Fix them for 1h with 2.5% glutaraldehyde solution. Change PBS solution into the bottle to immerse the clones and store them at 40C in the refrigerator. The cells that have received qi for four successive days are all treated as this. Then all these cells are fixed in 1% osmic acid for 1h, dehydrated in gradiant alcohol, imbedded and made into super thin sections for the observation under electronic microscope.
Observation with light microscope and computing the number of cells.
For four days since the inoculation, observe the cells everyday with light microscope. It can be seen that the cells in the qigong group are very bad at adhering to walls. They are sparse in distribution and there are many dead cells, while the cells in the control group grow exuberantly. They grow adhering to the cell wall and are densely distributed. The number of cells that become adhered to walls in the two groups is counted each day as follows (Table 1).
Statistical analysis shows P value is less than 0.05 (P＜0.05). The number of cells that get adhered to walls in the qigong group is less than that in the control group, as indicates that, with the effect of qigong, the growing trend of becoming adhered to walls in the cells is reduced and that qigong has an inhibiting and a killing effect on tumor cells growth. This effect makes the number of suspension cells increase, whereas the cells in the control group grow exuberantly and show a multiplication tendency.
Observe with transmission electronic microscope.
In cells Hela treated with Zhineng Qigong, there are not only melted and dead cells but also evident change in most of their super micro structure, as conforms to the features of apoptosis of cells. Observe them after the cells have been cultured at 370C for 24 hours. Chromatin of the cell nucleus agglutinates and tends
|Time||Qigong Group||Comparison Group||P Value|
|On the Day of Qi Transmission||2×104||2×104||0|
|At 24th hour after having been kept in culture||3.8×104||4.1×104||0.3×104|
|At 48th hour after having been kept in culture||6.9×104||8×104||1.1×104|
|At 72th hour after having been kept in culture||1.0×104||1.8×104||0.8×104|
Table 1 Number of Cells that Become Adhered to Walls in the Qigong Group and the Control Group (cell per bottle) tend to adhere to the boundary of the cell nucleus, ie., the phenomenon of chromatin going to the periphery. The cytoplasm condenses. The membrane of cell nucleus and that of the cytoplasm become rolled up (Fig. 1 ×6000, Fig. 2 ×8000). Having been cultured at 370C for 48 hours, it can be seen that the fragments of cell nucleus are enclosed by the membranes that have become projected on the cell surface and are separated into apoptotic bodies (Fig. 3 ×5000). Only few cells become dead in the control group when they have been cultured for 48 hours and 72 hours, which is shown in that the cell organ expand, not much change occurs in the external form of the cells and their chromatin slightly agglutinates (Fig.4 6000).
The effect that Zhineng Qigong brings about on the tumor cells cultured outside the human body shows that the therapeutic effect of Zhineng Qigong lies in external qi, ie., the external qi causes the above-mentioned change in the super micro structure of the cell without the qigong masters’ physical contact with the materials being treated and leads to the death of tumor cells, ie., the death of programmatic cells, or called the process of the apoptosis of cells. Meanwhile, from the number of cells, it can be seen that Zhineng Qigong external qi can inhibit the malignant growth of tumor cells and has a killing effect on them. It in a certain extent inhibits the feature of the tumor cell that limitlessly proliferates.
Apoptosis is a topic that gradually attracts more and more attention of researchers in recent years. It was first put forward by Kerr, an Australian scientist, in 1972, which provides people with further knowledge in the recognition of the death of cells. The death of cell includes the mechanism in two aspects: One is necrosis which happens when the cell is acutely damaged. The feature of the cell in this condition is that the cell swells, breaks with expansion and the substance contained in the cell spills, which leads to the inflammation in the surrounding tissues; while apoptosis belongs to “normal death” of the cell, the feature of which is as what has been described in Result in this article (See Fig. 1, Fig. 2 and Fig. 3).
Zhineng Qigong, when functioning on cells Hela, seems to be able to induce necrosis besides its ability to stimulate and cause the death of cell program. The two factors that bring about the death of cell combine to inhibit the infinite multiplication ability of tumor cells. Necrosis bases on how acute the damage in the cell is; while the death of cell program concerns the activation of some of the genes, eg., gene bcl-2, gene myc, gene P53, etc. and as well the influence of some apoptosis factors, such as Apo-1/Fas, TGFβ. Changes as these wait for further research in the days to come.
The programmatic death of cells may provide new strategies and new ways of thinking for human beings to get control over tumor. At the present, radiotherapy and chemotherapy are both established on the theory of cell virus. These methods have their disadvantages in overcoming drug-resistance and heterogeneity of tumor cells. Once people know about the detailed mechanism of adjusting and controlling the program of cell death, they can exert the special and super killing effect of gene induction on the tumor cells that vary in types and origins. Meanwhile, Zhineng Qigong induces apoptosis of the tumor cells and it shows that this “yuan qi” is actually material. Obviously, the mechanism of how Zhineng Qigong kills cancer cells still needs further exploration.
(1) Gao Cuihua, et al., an article waiting to be published in Oriental Qigong
(2) Cao Xuetao, an article published in Nature, 1989, 12:190
Zhang Jingbo: Professor
Liu Jiahong: Professor