| Background:Gastric cancer is one of the most common malignant tumors derived from epithelial tissues and poses a serious threat to human health.Due to specific dietary habits,climate and geographical location,according to the latest epidemiological survey,China has become one of the countries with high incidence of gastric cancer.China accounts for about 42% of the more than 1 million new cases of gastric cancer in the world each year.Of the approximately 800,000 deaths,China accounts for approximately 35%.Many factors have been reported to cause gastric cancer,including lifestyle,Helicobacter pylori infection,socioeconomic status,and environmental and genetic factors.Surgical resection is the main treatment for early gastric cancer,and conventional treatment for advanced gastric cancer is surgical resection combined with chemotherapy.However,chemotherapy drugs can inhibit tumor cell proliferation or promote tumor cell apoptosis,but also cause a certain degree of damage to normal tissue cells,resulting in a series of systemic toxic side effects.Therefore,the search for a treatment with good targeting and low adverse reactions has become a hot spot in the treatment of gastric cancer.Objective:By analyzing the inhibition of gastric cancer cell proliferation by comparing various intervention factors,the effects of combined docetaxel lipid microbubbles on the proliferation and apoptosis of human gastric cancer cells under ultrasound were preliminarily investigated.The protein level explores its specific mechanism of action to lay the foundation of the previous theory,and provides a theoretical basis for the subsequent development of more efficient loading of monoclonal antibodies targeting drug-loaded microbubbles.Methods:In this study,the docetaxel lipid microbubbles were prepared by mechanical oscillation method,and then the encapsulation efficiency,drug loading,preliminary stability study,drug release experiment and other ultrasonic drug-loading microbubbles were determined by ultraviolet spectrophotometer.Characterization after preparation.Based on the preparation of drug-loaded microbubbles,the effects of combined docetaxel lipid microbubbles on the growth of gastric cancer cell linesunder ultrasound-targeted rupture were further investigated.The study included the following four groups: a blank control group,a simple docetaxel group(DOC),a docetaxel microbubble group(DLLM),and a docetaxel microbubble + sonication group(DLLM+UTMD).The evaluation methods for inhibiting the proliferation of gastric cancer cells after treatment with intervention factors include: MTT assay for cell growth,detection of proliferation of human gastric cancer cells by incorporation,detection of apoptosis,and cell cycle detection by flow cytometry Distribution,mitochondrial membrane potential(MMP)detection,Western blot analysis.Results:1.The characteristics of docetaxel-loaded ultrasonic microbubbles were measured:(1)General characteristics: a circular vacuole with uniform size and good dispersion,and the concentration of drug-loaded microbubbles counted by the blood cell counting plate was 2.3×109--3.0 X 109 / ml,the particle size ranged from 475.8to 703.8 nm,and the average particle diameter was 621.4 nm.(2)Encapsulation efficiency and drug loading: The encapsulation efficiency of the free drug and microbubble was(73.04±4.76)%,(70.89±3.71)%,and the drug loading was 18.32±.1.1)%,(17.94±0.89)%,there was no statistically significant difference in the encapsulation efficiency and drug loading between the free drug volume and the microbubble drug dosage(P>0.05).(3)Drug release experiment: The drug concentration in PBS solution is extremely low before ultrasonic irradiation,but after ultrasonic irradiation,the drug concentration in PBS solution can be increased by 3times compared with that before ultrasound irradiation.(4)Stability study: The concentration of microbubbles and the size of the morphology did not change significantly within 7 days after being placed at 4 °C,but the concentration of microbubbles decreased slightly and the morphology gradually changed with the extension of the standing time.The encapsulation efficiency of the large and encapsulated ratios is lower than that of the freshly prepared microbubbles.After 60 Co ? ray sterilization,there was no significant change in the concentration and shape of the microbubbles.2.Effect of DLLM in combination with UTMD on BGC-823 cell growth:(1)DOC inhibited the growth of BGC-823 in a dose-dependent manner and maximum inhibition was observed at a dose of 4 ?M.(2)Compared with DOC or DLLM treatment alone,DLLM+UTMD treatment significantly inhibited the growth of BGC-823 cells,whereas DOC and DLLM inhibited3.The effect of DLLM combined with UTMD on the cell cycle of BGC-823:(1)Compared with the control group,DOC,DLLM and DLLM+UTMD significantly reduced the proportion of cells in S phase and increased it in G2 phase.(2)Comparedwith treatment with DOC or DLLM alone,treatment with DLLM+UTMD further reduced the proportion of S phase cells and increased them in G2 phase,and no significant difference was observed between DOC and DLLM groups.(3)By analyzing the expression of BrdU incorporation and cell cycle regulatory protein,it was found that in the four experimental groups,BrdU was the lowest in the DLLM+UTMD group,while p53 and p21 were the highest.4.The effect of DLLM combined with UTMD on apoptosis of BGC-823 cells:(1)Compared with the control,DOC,DLLM and DLLM+UTMD significantly induced apoptosis of BGC-823 cells.(2)Apoptosis was further promoted by treatment with DLLM + UTMD compared to treatment with DOC or DLLM alone,whereas the levels of apoptosis in the DOC and DLLM treated groups were comparable.5.Effect of DLLM combined with UTMD on MGC in BGC-823 cells:(1)Treatment with DLLM+UTMD significantly reduced MMP levels in BGC-823 cells compared to treatment with DOC alone or DLLM.(2)The expression of Bcl2 was the lowest and the expression of Bax was the highest in the DLLM+UTMD group.Conclusions:This study found that compared with the DOC or DLM treatment group alone,The DLM plus UTMD treatment group can significantly inhibit the growth of the cultured gastric cancer cell line BGC-823 by preventing the G2/M cell cycle,inhibiting cell DNA synthesis,promoting apoptosis,and destroying mitochondrial membrane potentials.In addition,the expression of Bcl2 was found to be significantly reduced in the DLM + UTMD group,while the expression of p53,p21 and Bax was significantly increased.Therefore,DLM + UTMD therapy is more effective in inhibiting gastric cancer cell proliferation and inducing apoptosis than DOC or DLM alone,which shows that DLM + UTMD can be used as a new strategy for gastric cancer treatment. |
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