| Conjugated linoleic acid is one of polyunsaturated fatty acids with the special physiological activity. In recent years, some research has confirmed that CLA has a lot of benifit for human health, such as anti-cancer, curing atherosclerosis and losing weight. Animal experiments and tumor cell culture experiments show that, CLA can prevent colon cancer. In the process of digestion and absorption, the activity of CLA is affected by the stomach low pH value environment, at the same time reduce its activity to inhibit colon cancer LoVo cell.In the research we choose (3-LG as a carrier material to protect CLA physiological activity. P-LG secreted by the breast epithelial cells, which is the important composition of milk cow whey protein and is peculiar to milk. β-LG as a member of the Lipocalin protein family, its protein molecules structure has a special calyx structure which can combine with the small hydrophobic molecules, and is not easily digested and decomposed in the low pH in stomach. This function make it become an ideal antitumor drugs which can used to oral colon-specific drug delivery system.There were total4studies in this paper:(1)β-LG-CLA was preparated and its property was analysised. Heat treatment and Titration were used to combine β-LG with CLA successively and P-LG-CLA complex was prepared. For β-LG-CLA complex in the gastrointestinal tract with low pH, the change of particle size and ζ potential were measured by the Delsa Nano Czeta (ζ) potential and particle size analyzer, its stability in gastrointestinal condition is also investigated under the function of digestive enzymes, pepsin and trypsin, and the result was showed by SDS-PAGE electrophoresis. These studies were used to explore the stability of P-LG-CLA complex molecular structure in the process of targeted intestinal.(2)The morphological observation of LoVo cells:In the inverted microscope, we tested the morphological changes of LoVo cells which were used to different concentration of p-LG-CLA complex, and preliminary judge whether the drug had influence on LoVo cell proliferation.(3)The effect of β-LG-CLA complex on inhibiting proliferation and inducing apoptosis for LoVo cells:β-LG-CLA complex inhibits proliferation of LoVo cells detected by WST-1assay; the changes of morphology was detected by Inverted microscope; the apoptotic alterations of LoVo cell in morphology was detected by Hoechst33258staining and examined by fluorescence microscope. Flow cytometric was used to detect the apoptosis rate of cell.(4)The study of mechanisms of apoptosis of LoVo cells promoted by β-LG-CLA complex stimulate:LoVo cells membrane electric potential were detected by JC-1fluorescent staining; the content and activity of protein caspase-3in LoVo cells were assayed with the Caspase-3Activity Assay Kit; Western blot was used to test Cyt C expression quantity change.Through the study, we get the following conclusion:(1)We added c9, t1l-CLA drop into P-LG solution being preheated to50℃, and successfully obtained the β-LG-CLA complex. The stability β-LG-CLA complex in gastrointestinal pH condition was detected using Delsa Nano C zeta (ζ) potential analyzer.The ζ-potential of β-LG-CLA complex reduced from40mV to16mV as the pH value increased from1to3. With the pH value increased from6.0to7.4, the zeta-potential of β-LG-CLA complex was all lower than-40mV, meanwhile the particle size of β-LG-CLA complex distributed in about170nm. Only slight decomposition occurred when β-LG-CLA complex under the function of digestive enzymes, pepsin and trypsin, and the result showed by SDS-PAGE.(2)Inhibition of proliferation was measured by WST-1assay, as a result, the inhibition rate of LoVo cell proliferation increased with the drug acting time increasing at the same drug concentration, and the inhibition rate of LoVo cell proliferation increased with the drug concentration increasing at the same acting time.(3)The morphological change of the LoVo cell cells were detected by the optical microscope and fluorescence microscope.In this study, we found that LoVo cell appeared typical apoptosis form when the cell were treated with different concentrations of β-LG-CLA complex. We found that there were significant difference between the different concentration of P-LG-CLA complex and a control group when flow cytometric was used to detect cell apoptosis rates.(4)The mitochondrial membrane potential showed different drop when LoVo cells were measured by JC-1fluorescent staining,meanwhile,caspase activity had different degrees of improvement and mitochondria Cyt C content had different degrees of decline.In this topic, P-LG had a particular chemical structure named calyx which could combine with small hydrophobic molecules, the characteristics were innovatively used to synthesis of β-LG-CLA complex which could be applied to oral colon-specific drug delivery system. P-LG-CLA complex and the carrier of β-LG didn’t have poisonous effect themselves, meanwhile it had been confirmed that β-LG-CLA complex was basically stable under the gastrointestinal condition in vitro, which showed thst it was a perfect application prospect at the aspect of targeting intestinal tumor therapy. This topic confirmed that different concentration of β-LG-CLA complex could inhibited the proliferation of LoVo cell and promoted the apoptosis of cell, the mechanism of its action may be that β-LG-CLA complex stimulate the changed of mitochondrial membrane and the stability of mitochondrial membrane,which could lead to Cyt C released from mitochondria to Cytosol,and further activate Caspase-3, ultimately leading to cell death. The combination of P-LG and CLA opened up new way for the diagnosis and treatment of cancer and provided a new idea for the development of functional food as a health drugs. |
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