Team:JSNU-China/Description
Background
Although it is one of the most researched and funded fields in medicine, cancer is still a major cause of morbidity and mortality worldwide, with 14 million new cases and over 8 million deaths per year. It is the second cause of death worldwide, and it’s responsible for quarter of the death cases among developed countries.
People try many ways to keep health or prevent cancer spread, including take some kinds of food, which contain more anthocyanin. Even, during cancer threapy, some doctors also encourage patients to use anthocyanin-like drug .
But，in fact, we can’t destroy these bad boys-cancer cell s in our body or just keep away cancer easily, using own AK47 -anthocyanin.
About anthocyanin
Citrus fruits and vegetables contain much anthocyan which is a naturally flavonoid with powerful anticancer function. Through the protection of genetic material(DNA), anthocyanins will be able to indirectly protect us against cancer. Although the long-term, but anthocyanins do have an indirect protective effect. The anthocyanins free radical scavenging effect, can also make the cancer cells can not be successfully diffused a way to protect healthy cells from being more aggressive cancer. On the other hand some cancer formation through the material was dissolved tumor tissues and cells, these cancer cells dissolve meat on the back of an animal protein and meat on the back of an animal, and anthocyanins protect the protein from the impact of meat on the back of an animal protein.
About KLF4
Fistly, Kruppel-like-factor 4（KLF4）is a zinc finger type transcription factor known to be highly expressed in various human tissues, including epithelial cells of gastrointestinal-tract.Secondly, KLF4 is able to bi-directionally regulate genes that are controlling cell cycle regulation, epithelial differentiation, EMT, and apotosis.Thirdly,KLF4 is downregulated in most kinds of cancer.
Experience
Preliminary experiments
We first examined the various cancer cells in the expression of KLF4‘s .
(a)Q-PCR
(b)Western blot
Through these two methods, we examined the KLF4 expression of two aspects, about RNA and protein levels. KLF4 is indeed a lower expression in gastric cancer cells.
2)At the same time we treated with different concentrations of anthocyanin GES1 and HGC27 cells, as a control. Illustrates the anthocyanins on gastric cancer cell is certain killing effect, and is not too much of a role for normal cells.
3)Before constructing plasmids , we transfect SUM52 with GFP plasmid to check transfection efficiency. The results as shown:
The experimental results show that our transfection is efficient.
Then, we use the concentration of 0 ug/ml, respectively 100 ug/ml, 200 ug/ml, 300 ug/ml anthocyanins handled transfection GFP plasmid SUM52 cells, then the cell apoptosis by fluorescence microscope observation.The results are as follows.
If the expression of klf4 is higher, will the cancer cells die faster after dealing with anthocyanins?
Formal experiment
We decided to construct KLF4 over-expressed plasmid based on the results of preliminary experiments.
Through PCR, restriction enzyme digestion, connections, repeated tests have been completed the construction of the plasmid. Then, we put KLF4 over-expression plasmid into 293 cells , and spent a long time to get stably expressing screened cell lines. Then we do western blot again detection of protein inside cells, protein expression plasmid construction is proof of our success.
We successfully constructed KLF4 over-expression n of cell.
According to the results of preliminary experiments, the cancer cells have a certain anthocyanins cytotoxicity .Then we dealt with the cells of anthocyanins.
and then every day CCK8 reagent for detecting cell viability, We also treated with the same concentration of anthocyanins normal 293 cells were used as controls
So We have two ways to check the viability of cells - electron micrographs of the cells and the data detection CCK8, we have two sets of data.
Two sets of data show, KLF4 anthocyanin treated with high-expressing cells on the first day of the death, and the third day all died. While the normal 293 anthocyanins cells treated in four days there has been death, by comparison, KLF4 over-expression greatly increased the sensitivity of cancer cells anthocyanins.
Thus, we conclude that KLF4 over-expression greatly increased the sensitivity of cancer cells anthocyanins. Then, we have great interest about how KLF4 regulatory anthocyanins and cancer cells generated, we decided to study the issue of mechanisms.
Future Work
According to KLF4 structure, We construct to build another four mutant.
However, due to time constraints, we have not had time to make this part of the work, which will be our next will try to complete the work
Protocal
The experimental operation we mainly used
1.EnoGeneCell Counting Kit-8
Detection principle
◆Cell Counting Kit Acronym CCK8 kit is based WST-8 (chemical name: 2- (2-methoxy-4-nitro-phenyl) -3- (4-nitro-phenyl) -5- (2 , 4-sulfophenyl) -2H- tetrazolium monosodium salt) is widely used in cell proliferation and cytotoxicity of fast, high-sensitivity detection kit.
◆WST-8 belonging to MTT upgrade products, works as follows: In the case of electronic coupling agent is present and can be restored Dehydrogenase mitochondria produce highly water-soluble orange formazan product (formazan). Color depth and proliferation of cells is directly proportional to cell toxicity. OD value was measured using a microplate reader at a wavelength in the 450mM indirectly reflect the number of viable cells.
◆CCK8 method is widely used, such as drug screening, cell proliferation assay, cytotoxicity assays, tumor susceptibility testing and detection of biological activity and other factors.
Cell Viability Assay
1, the cell suspension seeded in 96-well plates (100μL / well). The plates were pre-incubated in an incubator (37 ℃, 5% CO2).
2, was added to each well 10 μL CCK8 solution (be careful not to bubble Kong Zhongsheng, they can affect the reading OD values).
3, the plates were incubated in the incubator for 1-4 hours.
4, then determined by comparing the absorbance at 450nm.
5, if the OD value temporarily, can be added to 10 μL 0.1M HCL solution or 1% w / v SDS solution to each well, and the cover plates stored in the dark at room temperature. Within 24 hours measured absorbance change does not occur.
Cell viability * (%) = [A (dosing) -A (Blank)] / [A (0 dosing) -A (Blank)] × 100
2.Molecular Cloning
3.Western blot
Extract proteins
1.1000r/min centrifuge for 5 minutes to remove PBS.
2.Add lysis buffer and shake it.
3.12000r/min freezing centrifuge for 15minutes
4.Add loading buffer and boil for 10 minutes
5.Put it on the ice.
SDS-PAGE
1. Plates:The sealed box with silica gel placed flat on the glass, and then the concave glass and flat glass overlap, the two glass stand up to make contact with the bottom of the desktop, two glass hand clamped into the electrophoresis tank, then insert Xiecha board to moderate degree, you can glue.
2. Polymer gel:Separating gel and stacking gel preparation: The following table sequentially and proportion solution, configuration of 10% separating gel and 4.8% stacking gel.
3.
| Separating gel | Stacking gel | |
| ddH2O | 4.95ml | 3.05ml |
| Arc-Bis(30%) | 6ml | 0.67ml |
| Tris-Hcl | 3.75ml(PH=8.8) | 1.25ml(PH=6.8) |
| 10%SDS | 150ul | 50ul |
| 10%APS | 150ul | 50ul |
| TEMED | 15ul | 10ul |
Following the above table, mix them.After separating gel formulation, add it to the gel, be careful not to produce bubbles. Then add water carefully to make surface covered with water.Put them on room temperature about 30-40min. When absorb the water completely, prepare stacking gel according to the above table. Add stacking gel and insert the comb .Finally,carefully remove the sample.
4.Loading:add sample to the sample tank
5.Electrophoresis
Transfer and block
1.transfer the protein to PVDF membrane.
2.block the membrane with 5% milk 1h.
3.add the first antibody and overnight at 4 degrees temperature.
4.wash membrane 15min 3 times.
5.add second antibody 1h at room temperature.
6.wash membrane 15min 3 times.
Expose
Expose and we can see the result clearly.
4.Cell culture
Cell culture technology refers to the number of cells after a lot of training to become a simple single cell or a little more differentiated cells, which are essential aspects of cloning technology. We can get a lot of cells or metabolites through cell culture. Because biological products are derived from the cells, it can be said cell culture technology is biotechnology core, the most basic technology.
Cell recovery
1.We put cryopreserved tubes in 37 degrees temperature water bath pot and shake it until dissolved.We can complete the thawing about one to two minutes.
2.Put cryopreservation solution and 5 ml of culture into a centrifuge tube and Pipet.
3.1000r/min centrifuge for 5 minutes
4.Add the precipitate to complete medium, pipetting, transferred to a Petri dish.
Cell culture
Add 55ml serum and 6ml Dual anti to the medium.
Collect cell
6.Add Trypsin and stop digestionafter cell culture medium plus curled .
7.Absorb the culture medium to completely blow down cells.
8.Transfer the cells to a centrifuge tube and centrifuge for 5 minutes
9.Discard the supernatant, add wash cells with PBS.
10.1000r/min centrifuge for 5 minutes
11.Discard the supernatant and add frozen -80 degrees to cryopreservate.
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Jiangsu Normal University, 101 Shanghai Rd, Tongshan District, Xuzhou, China
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