| Colorectal cancer is a common malignant tumor of digestive tract. The incidenceof colorectal cancer occupies second place in malignant tumor in westerncountries,and fourth place in our country. The5years survival rate is only50%, itmeans about half of the postoperative patients will finally happened to recurrence andmetastasis, especially of liver metastasis. Exploration of the new method in thetreatment of colorectal cancer has high clinical value. The research progress ofcolorectal cancer stem cells in isolation and its biological characters adds us newresearch direction for the treatment of colorectal cancer.Hedgehog (Hh) signaling pathway was discovered in1980, its disordersassociated with tumor development.. Members of Hh pathway abnormal geneexpressiong may cause downstream target genes transcription abnormalities,associated with abnormal cell division and proliferation, which ultimately can lead totumor formation. Cyclopamine,as Hh signal pathway inhibitors, can induced cancercell apoptosis at the same time,,rarely affects the growth of normal cells.In2009,Warnat F study found that Hh pathway can promote colorectal cancercell growth and metastasis, Hh pathway activation are closely associated with thestem cell survival and self-renewal related.Stem cell is a kind of primitive cells with the ability of self-renewal andmultilineage differentiation potential. Researchers have Find the common pointsbetween malignant tumor and stem cell in the biological behavior in cell growth,metastasis and recurrence characteristics. Then some of them hypothesized that tumorstem cell differentiation proliferation disorders arising from abnormal tissue, and putforward put forward the tumor stem cell theory.The theory indicates that tumor is a kind of disease occurred at the stem cell level, tumor stem cells have stem cell-like properties, with self-renewal, unlimitedproliferation and multilineage differentiation capacity; although it accounts for only afew in the tumor tissue, but it is the source of tumor occurrence, development andmetastasis. The cancer stem cell theory, contribute to better understanding of tumororigin and essence.It changes the mode of treatment in tumor therapy. Targetedtherapy in the tumor stem cells provide a cure possible method in malignant tumor.In2006, Catherine and Lucia were published their research findings in thecolorectal cancer stem cell. They isolated CD133positive colorectal cancer cells inthe fresh tissue specimens suspension of colorectal cancer patients. Find itstumorigenic ability is much higher than that of without separation and sorting ofCD133negative cells in NOD/SCID mice, proven their stem cell characteristics.It is based on the current status in the treatment of colorectal cancer, colorectalcancer stem cell theory and practice development, colon carcinoma cells in thepresence of Hh pathway abnormalities, this paper put forward the research of " thestudy in effects of Hh pathway blockade on colorectal carcinoma cells and colorectalcancer stem cells".In the first part of this study, we applied the Hh pathway antagonist cyclopamineon colorectal cancer Caco-2cell for SMO level target block. the caco-2cells showedsignificant inhibition of proliferation, and confirmed that this effect is mainlyachieved through the promotion of apoptosis.Because mitochondria and apoptosis are closely related, this paper furtherapplication of proteomic technologies to Caco-2cell mitochondrial protein differenceanalysis, a total of57species found obvious differences in protein expression, itsfunctions mainly related to cell proliferation and apoptosis, cytoskeleton, stress andimmunity, metabolism of nucleic acid and protein synthesis, signal transductionrelated enzyme system.Which is related to cell proliferation and apoptosis related protein5, ATF6,Clusterin, OPA1, RGD1565411expression, Cofilin expression upregulation, furtherstudies on it, may deepen our understanding of the Hh pathway and Hh pathwayregulating downstream genes and understanding its interactions with other signal systems. The above research confirmed that the Hh pathway blockade on colorectalcancer with proliferation inhibition effect and its possible mechanisms.The second part of this paper, mainly on colorectal cancer cell sorting andidentification.We isolated primary colorectal cancer cells from malignant ascites of colorectalcancer,Application of NOD/SCID mice as tumor animal, Vaccination in primarycolorectal cancer cells, effectively forming tumor tissue.and these tumors can befurther amplified and isolated as a source of stem cells. By chromosome karyotypeanalysis, the colorectal cancer cells show a complex karyotype, consistent withcolorectal cancer cell line standard.Isolated cells need to confirm their stem cell characteristics. In vivo experiments,we conducted clone formation test wih the difference ability to tolerance inserum-free culture characteristics between cancer cells and cancer stem cells. In vitrowe conducted murine tumor experiment in NOD/SCID mice. simultaneousdetermination of their differentiation and CK20expression. the above three aspects ofthe experiment confirmed that the sorting of CD133positive colorectal cancer cells totumorigenic ability is stronger than before separation of colorectal cancer cells, whichpossess the stem cell property.In the third part of this paper, we test the effect of of Hh signaling pathwayinhibitor on colorectal cancer stem cells.Firstly, we conducted cell line proliferation inhibition experiment in the newcolorectal cancer sorting from the malignant ascites in colorectal cancer. found thatcyclopamine can effectively inhibit the growth of colorectal cancer cells. At the sametime, found the Hh channel blockers can make the colorectal cancer cell line ofCD133positive cells ratio decreases, preliminary confirmation the effectiveness ofcyclopamine on cultured human colorectal cancer cell line and CD133positive cells.On the whole, through the above study, we confirmed the Hh pathway blockadeof cyclopamine can effect inhibition of colorectal cancer cell line proliferation ofCaco-2, this inhibitory effect of main mechanism may promote cell apoptosis. At thesame time of cyclopamine promotion during apoptosis, mitochondrial alterations may have an important role in. Proteomic detection of differences in mitochondrialproteins including cell proliferation and apoptosis, cytoskeleton, stress and immunity,metabolism of nucleic acid and protein synthesis, signal transduction related enzymesand other more obvious changes and provide more clues for the Hh pathway effectgene research.We successfully amplified new colorectal cancer cell lines in colorectal cancerpatients with malignant ascites, chromosome analysis showed complex karyotype.Taking CD133as marker beads, by magnetic separation technology, two times ofcolumn separation, We can separate high purity of CD133positive cells. Separation ofCD133positive cells in vitro formation of cloning and in vitro tumor experiments toprove its tumorigenic ability was significantly higher than that of its sources forcolorectal cancer cell populations, the differentiation of epithelial markers increase,thus confirming the possessing the properties of stem cells.Through the Hh pathway blockade effect in the colorectal cancer cell lines, wefound the proliferation inhibition effect,of cyclopamine on this cell line, but ist effectslightly weaker than on Caco-2proliferation inhibition. At the same time, Hh pathwayinhibitor cyclopamine reduces cell population of CD133positive cells ratio. In vitrotests have found it can inhibit the growth of colorectal cancer stem cell colonyforming ability and migration ability, in vivo experiments found that can reducecolorectal cancer stem cells to the tumorigenic ability. Confirmed that the Hh pathwayblockers can reduce the ability of colorectal cancer stem cells to tumorigenic.In summary, we think Hh channel blockers can inhibit malignant biologicalcapacity on colorectal cancer and colorectal cancer stem cells. The further study onthe mechanism will contribute to colorectal cancer targeted therapy of colorectalcancer, improve clinical curative effect. |
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