| Background Chloride ion, iodide ion and bromide ion, etc., anions(halogen) are important components that maintain intracellular environment and develop normal functions of humans. Dynamic equilibrium of water and various ions across the plasma membrane depend on the role of various channel proteins and transporters in the cytomembrane, while transmembrane transport of anions participates in all kinds of biological activity processes, including volume regulation, migration, proliferation, apoptosis, internal and external environment p H regulation of cells. Particularly, in various pathological damage processes, when changing internal and external osmotic pressure, such as cellular swelling, anions complete exchange inside and outside the cytomembrane by means of ion channels and transporters which play an important regulatory role. At the earlier stage, our group found that anion channels participated in the apoptosis process by constructing a myocardial apoptosis model through mitochondria, receptor and endoplasmic reticulum stress by staurosporine, ischemia reperfusion and tunicamycin separately. Applying specific inhibitor DCPIB or non-specific inhibitor DIDS can restrain death caused by cell apoptosis. Particularly in systemic heart, it can reduce infarct size, improve cardiac function and restrain occurrence of arrhythmia and play a role on protecting damaged heart. However, the application of anion channel blockers is only remained in the laboratory stage. Moreover, only a few kinds of drugs are in use and all monopolized by foreign reagent companies. For this reason, we aim at High Throughput Screening technology to find out compounds with proprietary intellectual property rights in the known natural compound library, while how to look for an efficient, low-cost and natural compound screening technology is extremely urgent and important. In order to look for an efficient, convenient and low-cost method for screening out natural compound, the researcher showed that fluorescent changes of yellow fluorescent protein can be regarded as a signal to display changes of anion concentration in cells. Enhanced yellow fluorescent protein(EYFP) is widely used for studying anion changes of secretory cells, nerve cells and tumor cells in digestive tract in recent years, because of higher p Ka value, more sensitive to halide, and being quenched by I-. The study plans to transfect the lentiviral vector carrying EYFP mutant protein(EYFP-H148Q/I152L) gene built by gene recombination technology into He La cells, establish stable cell models to regard as screening model of anion(halogen) channel blocker, apply constructed EYFP-H148Q/I152L-He La cell screening model, conduct high flux screening and evaluation on numerous natural compounds.Objective 1. To construct He La cells of stable expression mutation fluorescent protein EYFP(EYFP-H148Q/I152L) as the cell model of anion(halogen) channel blocker screening.2. Apply EYFP-H148Q/I152L-He La screen screening model to do preliminary screening on natural compound and evaluate the model.Methods 1. Gene recombination technology builds expressive EYFP mutant protein(EYFP-H148Q/ I152L) and puromycin-resistant lentiviral vector. 2. Lentiviral vector and plasmid mixture are amplified in 293 T cell and then He La cells will be infected by the obtained lentivirus. 3. Puromycin are added to screen out and purify target cells,to construct He La cells with a stable expression of mutation fluorescent protein EYFP(EYFP- H148Q/I152L). 4. Real-time quantification PCR and Western Blot are used to detect the over-expression of target gene EYFP-H148Q/I152 L in target cells.And passage the cells. 5. FLIPR TETRA real-time high flux fluorescence imaging analysis meter is applied to screen out natural compound. 6. The experiment can be divided into 4 groups: hypotonic I- solution is negative control group. Iso-osmia Cl- is positive control group. Hypotonic I- solution with DIDS group is the standard reference group, and hypotonic I- solution with compound group is the experimental group. 7. High flux screening model formula is evaluated by: Signal to Noise Ratio(Signal/ Background Ratio, S/B) = mean signal/mean background; Z's factor =(1-3×signal standard deviation + 3×background standard deviation)/| signal mean- background mean|. 8. Processing formula of high flux screening activity data for natural compound is shown as follows: Blocking compound reaction rate =(detected compound value-positive control value)/(negative control value-positive control value) × 100%; blocking activity=1- blocking compound reaction rate.Results 1. The mutation sequences EYFP-H148Q/I152 L were inserted into lentiviral vector and were proved to be right by using gene sequencing technology. 2. Lentivirus particles can infect He La cells successfully and the cells were puried by puromycin, as well as monoclonal proliferation. 3. Real Time-PCR and Western Blot detection displayed that target gene was overexpressed in He La cells(P<0.05, n=8). 4. Under fluorescence microscope, it can be observed that EYFP-H148Q/I152L-He La stable cell strain expressed proper yellow fluorescence. The efficiency is close to 100%. 5. Negative control group(hypotonic I- solution) showed obvious fluorescence quenching for cells; Iso-osmia I- solution group showed slight fluorescence quenching for cells. Both of them have significant differences(P<0.01, n=16). 6. Positive control group(iso-osmia Cl- solution) showed no fluorescence quenching for cells. By making a comparison between negative control group and positive control group, fluorescence intensity in both groups has the significant difference(P<0.01, n=16). 7. Standard reference group(hypotonic I- solution with DIDS group) showed weak fluorescence quenching. Compared with negative control group, fluorescence intensity in both groups has the signifiancant difference(P<0.01, n=16). 8. Concentration gradient experiment was conducted on standard reference drug DIDS. Finally, 500?mol/L and 50% of blocking efficient standards are regarded as the screening standard. By inoculating different cell density and different inoculation time, optimal experimental conditions can be obtained: 25000/100?L cell dnsity/ optimal fluorescence value of paving for 24 h. 9. Based on iso-osmia Na Cl group and hypotonic Na I group, high flux screening meter calculates Z' factor and Signal to Noise Ratio(SNR) to evaluate screening model. Through formula calculation, Z' factor is 0.53. SNR is 5.14, showing that sensitivity and specificity of the cell model conform to high flux screening standards. 10. Under the above-mentioned experimental conditions, by setting up negative, positive and standard reference group, fluorescence intensity of 6988 natural compounds is compared. The first screening acquires 425 kinds of positive effects. In order to further improve positive rate. The second repeated screening on the above-mentioned 425 compounds is conducted to acquire 200 positive results. The third repeated screening on the above-mentioned 200 compounds is conducted to acquire 7 positive compounds.Conclusions 1. Stable expressive EYFP-H148Q/I152L-Hela cell strain is constructed successfully. I-hypotonic stimulation displayed obvious fluorescence quenching. It is a stable, efficient and ideal anion(halogen) channel blocker screening model. 2. The optimal experimental concentration and judgment standards of standard reference drug DIDS are confirmed. 500?mol/L and 50% of blocking efficient standards are regarded as the screening standards of natural compounds. 3. High throughput screening standard formula can calculate Z' factor and Signal to Noise Ratio(SNR) to evaluate the good sensitivity and specificity of the model. It conforms to high throughputscreening standards. 4. High throughput screening is conducted for compounds with three times of positive results. 7 candidate compounds are acquired preliminarily from 6988 natural compounds. |
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