Jikai Gene Traditional Chinese Medicine Functional Gene Research Program (Tumor Direction)
I want to do Chinese medicine research, what can you do for me?
Ji Bo has a knot in his heart. When he was working in Fudan, he helped a clinician to see the subject. The ending was to take the two countries' natural faces. Is this ending very good? If it is good, it is not the knot in Ji Bo’s heart. Because, due to limitations of experimental techniques, there were no articles with more than 5 points.
In doing research, there are two things. One is that after I ask a scientific question, I know how to do it. The other is how do I do it. "Know" and " Do it . " In fact, the "feasibility analysis" in the country's nature is for the experts to see, in fact, it is for us to see. If a subject only knows how to do it, but can't "do it", what is the use of clinical disease research? Jibo keeps sharing high-scoring articles and wants to let everyone know how to do clinical research. And what you want to say today is to let you "do it."
The Chinese quintessence of Chinese medicine, I look forward to rejuvenating my generation! ! ! Jibo has a good wish to let more people understand Chinese medicine research by sharing high-scoring articles, and then interested in Chinese medicine research. Together with helping everyone "do it", it will promote the research of traditional Chinese medicine and accelerate the modernization of traditional Chinese medicine.
In the study of traditional Chinese medicine, the road to Jane, summed up one sentence is " drug E through the regulation of A gene affects B signaling pathway to inhibit the occurrence and development of C disease ." Different diseases have a slightly different way of studying. Take tumors as an example today. The three biggest threats to human health are cardio-cerebral vascular disease and cancer. Why don’t you take the first example? Because today’s saying is “do itâ€, from the perspective of feasibility, the research of cancer, from the technical level, even if you are in a remote mountainous area, as long as there is a simple molecular cell laboratory, you can carry out It is.
The disease of C is determined by our researchers. The drug E is also determined by our researchers. So what to study, the most important thing is the A gene. A gene that functions downstream of a drug, that is, a drug target. Therefore, our plan is to find the drug target of this A gene-drug.
Jikai Gene Traditional Chinese Medicine Functional Gene Research Program (Tumor Direction)
PS: Although it is the direction of the tumor, in fact other diseases are similar.
First, determine the cell model and drug function
1 , disease cell model
In fact, in the design of conventional topics, Ji Bo said that "the first step in scientific research is to determine the cell model and detection indicators." Why don't you say that the test indicators are determined here, because it is needless to say that drug treatment is the ultimate goal of killing tumor cells. The detection index here is to inhibit proliferation and promote apoptosis .
The cell's requirement is that a, a lentiviral infection can be performed. The best tool for the current operation of genes in cells is lentivirus-mediated. So from the very beginning, the cells must be able to perform lentiviral operations. b, cells can be screened for high-throughput function. To add this, because of this program, it is necessary to ensure that a positive result can be obtained. A good way to ensure a positive result is through high-throughput screening (as well as the screening platforms used by international pharmaceutical companies). Therefore, high requirements, cells must be able to perform high-throughput functional screening.
In the step of doing a tumor research, you don't have to worry about the unused tool cells. Jikai has more than 300 cell lines, and most of the tumors have tool cells that meet these two conditions.
2 , drug function
a. Detect IC50 on the tool cells. Many teachers said that I have done it myself and I don't have to. In fact, the IC50 is different for different cells of the same drug. Highly required, IC50 testing must be performed on subsequent tool cells.
b. According to the IC50 obtained in step a, one concentration is taken at the upper and lower sides of the IC50 concentration (plus the control is 3 groups), and the cell proliferation is inhibited by the CCK8 detection drug for 4 days. This step demonstrates that the drug can inhibit cell proliferation from drug concentration gradients and time gradients.
Second, drug downstream differential gene screening
Many teachers said that the drug is effective on the cell, looking for the drug downstream gene, dosing and negative control to make a whole genome expression profile chip. Haha. You are right. However, only a part of it.
1 , differential gene chip screening
The drug-added group and the negative control group were subjected to whole-gene expression profiling chips to find genes that were changed after drug treatment. Here is the 4V4 chip. Do you have 4 statistics for the group, and 3 are not statistically significant? Everyone notices that high requirements, because we are doing drugs, the group may be biased, and once the group is biased, as the foreigner said, "rubbish in, rubbish out", the data is basically useless. In order to ensure that there is something that can be done, there are four repetitions in the group. In the data analysis, if there is a sample with large deviation, the sample data is removed, and three reliable duplicate data in the group are entered into the downstream statistical analysis.
2 , differential gene and disease correlation analysis
Many people have questions in this step. For example, I am doing lung cancer, and the A gene has been reported in liver cancer, but it has not been reported in lung cancer. This gene is not good. In fact, for high-demand drug research, the principle is one, not reported in lung cancer. If it has been reported in liver cancer, I will focus on the relationship between drugs and genes (refer to the 6-point essay "Which broccoli can really prevent cancer?"). If the gene has not been reported in other tumors, I will focus on the relationship between the first gene and the tumor (see the 6-point essay in "Two broccoli can really prevent cancer").
From the analyzed genes, 30 genes that decreased after dosing were selected. Choosing a decline for å•¥, because the cells die after dosing, the gene declines, I later use RNAi to artificially let the gene decline, if the cells are also dead, indicating that we are looking for the right gene. However, if you want to do a drug up-regulation gene, you can also use the high-throughput function screening, gene manipulation tools can also use cas9-SAM to express high-throughput function screening. However, the risk is greater and you have more money.
Third, candidate gene function screening
1 , candidate gene drug correlation verification
The selected 30 genes were detected by QPCR technique between the drug-added cells and the negative control cells. Make sure that the genes that enter the follow-up are the genes that decline after dosing. Here, 20 genes are selected to enter the downstream.
2 , candidate gene manipulation tool preparation
The genetic manipulation tool here is lentiviral-mediated RNA interference. More than 3 fragments were designed for each gene, and 3 fragments were mixed and packaged into lentivirus (MIX). RNAi-MIX goes downstream.
Some teachers asked why they did not use the effective fragments of these 20 genes after they were detected. This is the difference between a single genetic study and a large-scale screening. We are also learning from foreign big pharmaceutical companies. The goal of large-scale screening is to narrow the scope, and the quick and economical solution is optimal. Then narrow down the scope, and then refine the details. In general, we screen 20 genes, the positive rate is 10%, and we will get 2 positive genes. If I started by screening out the valid fragments of 20 genes, you can calculate the cost and time required. Compared with who, I only need to do the effective fragment verification of these two genes, which is faster and more economical. (Amount, this is not a trade secret). Note that RNA interference was mentioned before commercialization of services without the Nobel Prize. A gene design has three targets, at least one of which is valid. Therefore, MIX is the best choice for high-throughput screening.
The Jikai gene now has ready-made libraries, including RNA interference libraries, Cas9 libraries (knockout and overexpression), and overexpression libraries. Achieve high-throughput functional screening requirements on tools.
3. High-throughput function screening
Core steps. In this step, we obtained a gene that inhibited cell proliferation after MIX interference. In summary of this gene, a, regulated by drugs, decreased after dosing. b. Cell proliferation is inhibited after down-regulation with RNA interference. c. Not reported in this tumor. At this time we can call him a drug target.
4 , tool verification
Because of the two genes, I did it one by one. See which target is the most effective. Subsequent experiments use this clear and effective target.
Fourth, the drug target cell function is perfect
This gene is functional, and there is only one cytofunctional experiment in front. Here again, other cell functions are used to confirm the function of this gene.
CCK8, BrdU, apoptosis, caspase 3/7 activity detection, proliferation or apoptosis marker WB detection, colony formation, and the like. Both are cell functional experiments in the direction of proliferation. Everyone notices that Jikai Gene provides scientific research services with the aim of increasing your value. The cooperation plan is here, there is promise. The gene found in this experiment, at least 3 experiments were positive, it is OK. If not, return to the high-throughput function filter and rescreen. Because we have a tool library, there is a screening platform. Everything is to enhance your value.
Do you want to apply for a natural nature? Here, there are genes, and the relationship between genes and drugs and gene functions are also determined. There is no problem with the scientific nature of the subject. If you want to ensure the winning rate, it depends on the writing skills of your bid.
Want to send an article, it is not recommended, because this gene is a good seed. Now to publish, SCI has no problem, but the score is not high. It is recommended to raise this seed to a large size and raise a good price after raising it. Ah, no, sell a good score.
How to raise it. Amount, many people say that Ji Bo wrote something, and today he wrote enough. Do not say it. If you want to know how to raise this seed, please contact us.
Everything works hard to enhance your value.
The Chinese quintessence of Chinese medicine, I look forward to rejuvenating my generation! ! !
PS: I hope that friends who have more exchanges with Jibo in learning and experimentation can pay attention to Jikai Gene WeChat and reply to the word "Jibo".
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