Assessment Method Of The Dietary Factor’s Effect On Cancer With The In Vivo Capture Of Circulating Tumor Cells Based On A Vein Indwelling Needle

Dietary factors have various cancer inhibit mechanisms to cancers. According to recent researches, the number of circulating tumor cells(CTCs) in bloodstream has a close relation with the overall survival rate and prognosis effect of patients with cancer. So, assessment of dietary factor’s effect on CTCs will contribute to the assessment of dietary factor’s effect on cancer.A in vivo capture method of CTCs that applied in mouse tumor models was established, and then this method was applied in the assessment of dietary factor`s effect on CTCs. A in vivo capture method of CTCs was established based on a vein indwelling needle in our previous work. Here, we had successfully applied this method in mouse tumor models to capture its CTCs, then identified and counted the CTCs by immunofluorescence staining techniques. The number of CTCs captured in vivo will reflect different dietary factor’s effect on the CTC.In chapter one, detection methods of CTCs, research development of inhibitory effects on cancer of dietary factors, and a brief introduction of assessment methods of dietary factors` inhibitory effects on cancer were summarized. The in vivo capture methods of CTCs and research developments of inhibitory effects on cancer of several anticancer food extracts was emphasized.In chapter two, a in vivo capture method of CTCs based on indwelling needle was applied in mouse tumor model. Detection and counting was carried out by immunofluorescence staining techniques. The indwelling needle was coated with polydimethylsiloxane(PDMS) on its flexible pipe, then the PDMS was modified with anti-EpCAM antibodies. This functionalized indwelling needle can capture CTCs that express the EpCAM specifically in bloodstream. Puncture the vein with the functionalized indwelling needle and keep for hours, then elute the cells that absorbed on the functionalized indwelling needle. To identify and count CTCs, fixation, permeabilization, incubation with different fluorescently-labeled antibodies and staining with DAPI will be carried out. Comparison of detection effects of in vivo capture method based on indwelling needle and in vitro dection method based on immune magnetic microbeads was carried out. Different levels of cancer progress in mouse tumor model was established via different inoculating numbers of cancer cells, and their CTCs were detected by in vivo capture method. Elution buffer, basement of fixation and time of permeabilization were optimized during the immunofluorescence staining. Phosphate Buffered Saline(PBS) was chosen as the elution buffer, ELISA plate was chosen as the basement of fixation and the time of permeabilization was 25 minutes. In vivo capture method can capture CTCs at the seventh day after inoculation, which is much earlier than in vitro detect method. With the development of cancer progress, the number of CTCs showed a increasing trend relatively.In chapter three, the number of CTCs of different dietary factor groups were detected by the in vivo capture method. Mouse tumor model were given gavage of curcumin, procyanidine, β-sitosterol,perillyl alcohol and tea polyphenol that were proved to have inhibitory effect on cancer. At the same time, high-sugar diet and high-fat diet were carried out as unhealthy dietary patterns for comparison. The number of CTCs, combined with the volume of carcinoma in situ and development of metastatic carcinoma, assessment of dietary factor’s effect on cancer could be more comprehensive. The results showed that several dietary factors had outstanding inhibitory effects on the number of CTCs and other stages of cancer progress. On the contrary, unhealthy dietary patterns showed accelerative effects on cancer progress.