Esophageal squamous cell carcinoma (ESCC) is a serious health concern, especially for people who enjoy spicy foods, like hot pot. In our previous study, by analyzing pre-/post-operative blood samples from clinical ESCC patients, as well as esophageal cancer tissue and paracancerous tissue samples using metabolomics and DIA proteomics technologies, we screen out key fatty acid pathways and sensitive proteins related with fatty acid metabolism, and the imbalanced fatty acid intake leads to the insufficient-expression of key functional proteins, which are necessary in repairing esophageal epithelial cells damaged by hot and spicy stimuli, the level of functional proteins play key roles in inhibiting inflammation and malignant lesions in esophageal tissues. Thus, in our present project, we firstly construct an exogenous plasmid system and transfect it into Escherichia coli to express high-level of functional proteins, then culture damaged esophageal epithelial cells and embed the protein with natural nanomaterial, to prove the repairing effects of functional proteins in vitro. This method effectively solves a series of esophageal epithelial cells damages caused by hot and spicy stimuli, and eventually integrates into a containing tablet, which brings great hope for reducing the incidence of ESCC.

ESCC (Esophageal Squamous Cell Carcinoma) is a common tumor of the digestive tract, and about 300,000 people around the world suffer and lose their lives from esophageal cancer every year. Therefore, prevention of esophageal cancer is bound to be an important direction for future research against esophageal cancer.The aim of this project is to develop a kind of containing tablets, so as to achieve the effect of maximizing the prevention of esophageal cancer.

It mainly consists of a four-step solution:
1.SAMPLING 2.STRUCTURE 3.VALIDATE 4.PRODUCTION

We firstly used passaging of inflammatory factor-treated cells to extract relevant proteins, the and confirms the fatty acid metabolism-related signaling pathways and key protein factors through metabolomics and DIA proteomics sequencing and analysis.And secondly we constructed protein high expression vectors through genetic engineering, selects E. coli as the receptor cell, obtains functional proteins and related signaling factors and verifies their expression.Then we constructed a damage model (heat stress treatment of esophageal epithelial cells), using Wb method to comprehensively assess the level of damage repair.Finally we embedded and tested the obtained functional proteins using a slow-release system, and making the tablets.

ESCC Esophageal squamous cell carcinoma (ESCC) is a prevalent form of malignant tumor found in the gastrointestinal tract, originating from the epithelial cells in the mucous membranes of the esophagus. Early symptoms of ESCC are not obvious and are similar to those of acute upper respiratory tract infections caused by viruses or bacteria, and are therefore not easily noticed by patients, resulting in the vast majority of patients being in the middle to late stages of the infection when the disease is diagnosed.

Problems with Current ESCC Treatment

At present, there are three main types of treatment for ESCC:Surgery,radiotherapy and chemotherapy. The research in ESCC is mainly focused on clinical treatment and basic science. In terms of clinical treatment, surgical resection is the main treatment method, which can achieve better curative effect for patients with early ESCC. For advanced patients, due to the rapid progression of the disease, it may not be possible to completely remove the tumour by surgical means such as local excision or total esophagus to remove only part of the lesion.In addition to surgery,radiotherapy and chemotherapy are often used as adjuncts.But it generally shows the prognosis of adverse symptoms. These are why preventive measures against esophageal squamous cell carcinoma are necessary.

Our team members cooperated to complete cell transfer, agarose gel electrophoresis, WB, PCR and other experiments, and wrote experimental reports. We made posters, leaflets and questionnaires,and carried out offline publicity activities in Sanlitun. In order to improve the usability and rationality of chemical tablets, we interviewed business owners and relevant professional doctors, asking about the market prospects and medical efficacy of chemical tablets. In addition, we have completed the publication of many official accounts, and at the same time operate TikTok and Xiaohongshu accounts, and the construction and compilation of wiki web pages are also carried out methodically.

Experimental steps:
I. Run the gel

1.Prepare electrophoresis liquid: 500ml pure water + SDS buffer powder.
2.Put the electrophoresis plate on the electrophoresis tank (red corresponds to the positive pole and blue corresponds to the negative pole) and put it into the electrophoresis instrument.
3. Pour in the electrophoresis liquid to the scale line.
4.Absorb 20 microliters of protein samples and add them to the corresponding card slot of the electrophoresis board (repeat this step until all samples are added).
5.Absorb 15 microliters of protein upper liquid buffer and 5 microliters of marker, and add them to the electrophoresis board card slot.
6.Connect the power supply and run the glue for 40 minutes until the marker appears in the corresponding interval.

II. Transfer film
1.Use buffer (appropriate amount) to soak the filter paper*2 and black sponge net*2, and use methanol (appropriate amount) to soak the PVDF membrane*1.
2.Remove the colloid from the electrophoresis plate and cut it into the appropriate size according to the protein traces at the bottom of the colloid.
3.Stack the materials in order. After putting them into the colloid, you need to cut a triangular notch in the upper left corner of the PVDF film with scissors.
Stacking order: black splint, black sponge net*1, filter paper*1, PVDF membrane*1, colloid*1, filter paper*1, black sponge net*1, black splint.
4.Put it into the electrophoresis instrument.
5.Pour in methanol (soak the PVDF membrane) and pour in the electro-transfer liquid to the scale line 4gels.
6.Run the gel for 1 hour and 40 minutes.

III. Drain membrane
1.Prepare 5% skimmed milk powder solution (the purpose is to reduce the impact of other useless small molecule proteins on the final development).
2.Open the black splint and build the PVDF membrane to the appropriate size of the incubation box.
3.Remove the colloid.
4.Use tweezers to transfer the PVDF membrane (which already has traces) to the incubation box.
5.Pour in 5% skimmed milk powder solution to just cover the PVDF membrane.
6.Put the incubation box on the rocking bed and incubate for 1 hour.

IV. Incubate primary antibody (rat source)
1.Prepare primary antibody
2.Add 3 ml of one antibody to the incubation box and incubate for 2 hours.
3.Use a pipette gun to recover an antibody in the incubation box.
4.Use 1xTBST to wash 3 for 5 minutes.

V. Incubate second antibody (sheep source anti-rat)
1. Prepare second antibody.
2.Add the second antibody to the incubation box and soak the PVDF membrane (3ml-4ml).
3.Place the incubation box with two antibodies on the shaker and incubate for 1 hour.
4.Pour out the second antibody in the incubation box.
5.Use 1xTBST to clean 3 for 8 minutes.

VI. Fluorescent color rendering
1. Meso Scale Discovery
(1) Prepare solution, A liquid: B liquid = 1:1.
(2) Take out the PVFD film and put it in the equipment.
(3) Apply the prepared solution evenly on the PVDF membrane.
(4) Start the instrument to calculate the exposure time and wait for 42 seconds.
(5) Observe the image.

Polymerase chain reaction (PCR)
I. Experimental purpose: PCR is a widely used molecular biology technology. Its main purpose is to quickly and specifically amplify specific DNA fragments in vitro.
II. Experimental steps:
1. Prepare the reaction components
(1) Buffer
The standard buffer contains 1pmol/ml Tris · HCl, and its pH is 8.3-9.0 (room temperature), while at an extended temperature (72 ℃), the pH value is close to 7.2. The buffer contains a divalent cation, which is used to activate the active center of DNA polymerase, generally using Mg2+. (2) dNTP
The abbreviation of deoxyribose nucleoside triphosphate is a general term including dATP, dGTP, dTTP, dCTP, etc., that is, the material that synthesizes a new DNA chain. The concentration of 4 dNTPs is equal, and the total concentration is generally 200pmol/ml (i.e. saturated concentration). dNTP can be combined with Mg2+ to reduce the concentration of free Mg2+ and affect the activity of DNA polymerase. (3) Primer
The primer is a short single-stranded DNA fragment, which can be combined with complementary regions on the nucleic acid chain. Its function is to serve as the starting point of nucleotide polymerization, and DNA polymerase can synthesize a new nucleic acid chain from its 3' end. The length of the primer is generally 18-30bp. If it is too short, it will reduce the specificity. If it is too long, it will cause annealing between the primers and affect the effective amplification, and also increase the cost of primer synthesis. The base order of the primer cannot be homologous to the non-amplified region. (4) Template
The template is a single-chain or double-stranded DNA that provides the original information for PCR amplification. The purity of the template is low, but it cannot be mixed with protease, nuclease, DNA polymerase inhibitor, DNA binding protein, etc. Template DNA should be kept at a low temperature as much as possible. (5) Taq DNA polymerase
Taq DNA polymerase uses DNA as a replication template, from the 5' endpoint to the 3' end of the enzyme. The main activity of DNA polymerase is to catalyze the synthesis of DNA (with templates, primers, dNTP, etc.) and its complementary activities. It must always be stored at -20℃, containing glycerin. The optimum reaction temperature is 72 ℃, that is, the extended temperature. It is usually added at the end of the PCR system. 2. Mixed reaction components
(1) Inject ddH2O 11.5 microliters into the centrifuge tube twice with a pipettor.
(2) Add 1 microliter primer.
(3) Add PCR buffer 2 microliters.
(4) Add dNTP 2 microliters.
(5) Add the template (cDNA) 2 microliters.
(6) Finally, add 0.5 microliters of Taq enzyme.
3. Set the PCR program
(1) Denaturation: Raise the reaction temperature to 94-98°C, usually maintained for 30 seconds to 1 minute, so that the DNA double helix structure unspins into single-stranded DNA. (2) Annealing: Reduce the temperature to 50-65°C, usually for 30 seconds to 1 minute, so that the primer can bind to the single-stranded DNA template. (3) Extension: Raise the temperature to 72°C. At this temperature, DNA polymerase will begin to synthesize new DNA strands, extending complementary DNA sequences. 4. Steps 1 to 3 usually repeat 20 to 40 cycles to achieve exponential amplification of DNA fragments. 5. At the end of the last cycle, the temperature is usually kept at 72°C, the incompletely synthesized fragment is further extended, and then the reaction tube is cooled to 4℃.
III. Precautions:
1. Ensure that the concentration of each component is appropriate to avoid inhibiting the PCR reaction.
2. The primer design should be specific to avoid the formation of dimers or non-specific amplification.
3. The extraction and purification of PCR products may be a key step in subsequent analysis.

I. Experimental materials: agar sugar powder, TAE, comb, nucleic acid dye, electrophoresis tank, loading buffer, DNA standard molecular weight, sample, pipette.
II. Experimental steps:
1. Mix 1g agar sugar powder with TAE and mix it into 100ml gel solution (at this time, the agar powder will not be completely dissolved, the solution is still turbid, and there is precipitation at the bottom of the cup).
2. Heat the gel solution until the agar powder is completely dissolved in the TAE and the solution becomes transparent.
3. Put the comb on one side of the mold.
4. When the gel is cooled to 60℃, add 5-10 microliters of nucleic acid dye and shake it quickly.
5. Mix it well and pour it into the mold, and the thickness is about 0.5cm.
6. Wait 10-20 minutes for the glue to solidify.
7. Remove the gel and comb from the mold, immerse them into the TAE solution in the electrophoresis tank, and then pull out the comb.
8. Add the DNA standard molecule weight to one comb hole, and add loading buffer and samples to the other comb holes, each hole is 20 microliters.
9. Connect the power supply, run the electrophoresis and observe the results.


III. Precautions:
1. The tip of the pipette should be changed when absorbing different liquids.
2. When the sample is placed, wait for the tip of the pipette to leave the liquid level before the press is released.
3. When sampling, the pipette should be stretched vertically into the comb hole.

When making the product, we considered various product types such as oral sprays, powders, and tablets. However, after careful comparison, we chose tablets as the form of our product. Because compared to the first two products, tablets require the longest time for dissolution in mouth, allowing the product to act more comprehensively and for a longer period of time on the entire esophagus rather than directly entering the stomach. Additionally, tablets are more portable and easier to distribute in crowded and potentially harmful environments such as hot pot restaurants where esophageal injuries may occur. In terms of design, our product uses nanocapsule technology. Nanocapsule refers to the use of nanomaterials to encapsulate active substances into nanoscale particles. This allows our product to be accurately released in a esophagus and more efficiently act on the damaged area. As for the repair mechanism, our product repairs inflammatory cells by regulating and influencing the signaling pathways of the epithelial cells in the esophagus, ultimately preventing esophageal cancer.

Oleanolic acid (OA) nanoparticles were prepared by ultrasonic emulsification method, and the morphology was observed by SEM. Overexpressed MFG-E8 protein sample was successfully prepared into OA nanoparticles. Agilent 1290 liquid chromatography was used to detect encapsulated protein samples, with the following liquid phase conditions: Mobile phase: 0.5% formic acid water (A) - acetonitrile (B), flow rate of 0.3 mL/min Gradient elution conditions: 0-1 min, 30% B; 1-3 min, 30-80% B; 3-5 min，80-98% B; 5-6 min，98% B; 6-7.5 min, 98-30% B; 7.5-9.5 min, 30% B; 9.5 min, stop. Injection volume: 2 μL, column temperature: 35 ℃, detection wavelength: 350 nm. Production of chemical tablets: the concentration of OA embedded MFG-E8 protein was 1 μM, the accessory ingredient was natural peppermint extract.

Dong Xiaohong, et al."MFG-E8 Alleviates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Phagocytosing Myelin Debris and Promoting Remyelination.."Neuroscience bulletin 40.4(2023):483-499.

Hua Xiaomin, et al."Protective Effect of MFG-E8 on Necroptosis-Induced Intestinal Inflammation and Enteroendocrine Cell Function in Diabetes."Nutrients 14.3(2022):604-604. .