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The Study on the Expressions of 4-1BB and 4-1BBL in Different Gastric Cancer Cell Lines

Author: WangLei
Tutor: SongZhenChuan；LiYong
School: Hebei Medical University
Course: Surgery
Keywords: gastric cancer tumor immune 4-1BB 4-1BBL TNF-α IL-2 IL-10
CLC: R735.2
Type: Master's thesis
Year: 2009
Downloads: 64
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Abstract

Objective: Gastric cancer is one of the most common malignant tumors in the world. There is no any significant break through in its treatment,although the gastric cancer has been adopted multi-method therapies and its survival rate has risen.The one cause of difficult to cure relates to tumor immune escape.The human anti-tumor immune almost is cellular immunity by T cell mediated and the activation of T cell needs bisignal stimulation: the first signal is provided by the compound of TCR combined MHC; the second signal is provided by costimulatories on the APC and the corresponding ligands on T cell. Only when stimulated by two signals specific T cell can be activated to take cell immunity and kill tumor cell. A lot of studies show that tumor cell can escape body immune by absence of costimulatory.Besides CD28/B7, 4-1BB/4-1BBL is a pair of important costimulatory signal, 4-1BB is exists on the cell membrane superficial, and it expresses on activated T cell, monocyt, granulocyte and dendrite cell. 4-1BBL belongs to ultra- family of tumor necrosis factor (TNF). 4-1BBL is II form membraneglycoprotein,which exists on the cell membrane superficial,and it expresseson activated T cell and B cell, monocyt, and dendrite cell,neuroblastoma and some tumor cell. 4-1BB/4-1BBL play svarious roles on cell-to-cell interaction, cell adhesiveness, antigen present, T cell costimulation and signal transmission.Nowadays the study of costimulatory molecules 4-1BB/4-1BBL in gastric cancer is rare.In this study, We detect the gene and protein expression of 4-1BB and 4-1BBL in the different human gastric cancer cell lines, and detect the killing activity of lymphocytes against gastric cancer cell lines, we mixed lymphocytes and gastric cancer cell lines ,then test the level of IL-2、IL-10、TNF-αin the supernatant. We talk about relation with gastric cancer cell lines’immunity and costimulatory molecules 4-1BB/4-1BBL. We intend to farther study the mechanisms of tumor immune escape and to settle theory and experiment foundation of gene therapy in future.Methods: We select four different human gastric cancer cell lines (MKN-28、SGC-7901、BGC-823、MGC-803). We detect the gene and protein expression of 4-1BB and 4-1BBL in the different human gastric cancer cell lines, and detect the killing activity of lymphocytes against gastric cancer cell lines, we mixed lymphocytes and gastric cancer cell lines ,then test the level of IL-2、IL-10、TNF-αin the supernatant. All statistical analyses were carried out with SPSS 16.0 and P<0.05 were considered significant.Results:1 The results of 4-1BB mRNA expression in four different gastric cancer cell lines.After PCR amplification with 4-1BB and 4-1BBL specific primers,we could get specific straps of 414bp.With the gel image systemsemi-quantum analysis showed that the 4-1BB mRNA expressions were MGC-803(77.30%) , BGC-823 (71.68%) , SGC-7901(40.06%) , MKN-28(37.65%) in the sequence of from high to low.2 The results of 4-1BBL mRNA expression in four different gastric cancer cell lines.After PCR amplification with 4-1BBL specific primers,we could get specific straps of 465bp.With the gel image systemsemi-quantum analysis showed that the 4-1BBL mRNA expressions were MKN-28 (46.63%), SGC-7901 (37.13%), BGC-823 (16.43%),MGC-803(14.14%) in the sequence of from high to low.3 The results of 4-1BB protein expression in four different gastric cancer cell lines.All the four gastric cancer cell lines (MKN-28、SGC-7901、BGC-823、MGC-803) express 4-1BB protein. The protein of 4-1BB was found to be distributed in the cell membrane and in cytoplasm in all the four cell lines through techniques of cytoimmunochemistry staining. The 4-1BB protein expressions were MGC-803, BGC-823, SGC-7901,MKN-28 in the sequence of from high to low.4 The results of 4-1BBL protein expression in four different gastric cancer cell lines. All the four gastric cancer cell lines (MKN-28、SGC-7901、BGC-823、MGC-803) express 4-1BBL protein. The protein of 4-1BBL was found to be distributed in the cell membrane and in cytoplasm in all the four cell lines through techniques of cytoimmunochemistry staining. The 4-1BBL protein expressions were MKN-28, SGC-7901, BGC-823, MGC-803 in the sequence of from high to low.5 MTT method measure the killing activity of lymphocytes against gastric cancer cell linesAt 24 hours ,the killing activity effect from high to low: MKN-28, SGC-7901, BGC-823, MGC-803.E/T is 20:1and 10:1,BGC-823 compare with MGC-803have no covariance to learn meaning (P>0.05). Group compare with other group have covariance to learn meaning (P<0.05).At 48 hours, the killing activity effect from high to low: E/T is MKN-28, SGC-7901, BGC-823, MGC-803. E/T is 20:1and 10:1,BGC-823 compare with MGC-803have no covariance to learn meaning (P>0.05). MKN-28 compare with SGC-7901 Group have no covariance to learn meaning (P>0.05). Group compare with other group have covariance to learn meaning (P<0.05).At 72 hours, the killing activity effect from high to low: MKN-28, SGC-7901, BGC-823, MGC-803. E/T is 40:1, BGC-823 compare with MGC-803 have no covariance to learn meaning (P>0.05). Group compare with other group have covariance to learn meaning (P<0.05). 6 We mixed lymphocytes and gastric cancer cell lines ,then test the level of cell factor in the supernatant.6.1 The level of cell factor TNF-αsecreteTNF-αsecrete from high to low: MKN-28, SGC-7901, BGC-823, MGC-803.E/T is 40:1 and 10: 1 BGC-823 compare with MGC-803have no covariance to learn meaning (P>0.05). Group compare with other group have covariance to learn meaning (P<0.05).6.2 The level of cell factor IL-2 secreteIL-2 secrete from high to low: MKN-28, SGC-7901, BGC-823, MGC-803. E/T is 20:1 , BGC-823 compare with MGC-803 have no covariance to learn meaning (P>0.05). Group compare with other group have covariance to learn meaning (P<0.05).6.3 The level of cell factor IL-10 secreteIL-10 secrete from high to low: MGC-803,BGC-823, SGC-7901, MKN-28Group compare with other group have covariance to learn meaning (P<0.05).Conclusion:1 The four different gastric cancer cell lines all express 4-1 BB at the gene and protein level. MKN-28 and SGC-7901 express low level of 4-1 BB, MGC-803 and BGC-823 to express high level of 4-1 BB, explain that the expression level of 4-1 BB relate to gastric cancer cell lines of divide degree.2 The four different gastric cancer cell lines all express 4-1 BBL at the gene and protein level. MKN-28 and SGC-7901 express high level of 4-1 BBL, MGC-803 and BGC-823 express low level of 4-1BBL, explain that the expression level 4-1BBL relate to gastric cancer cell lines of divide degree.3 The killing activity of lymphocytes against gastric cancer cell lines increase with the 4-1BBL expression more high.Explain the killing activity of lymphocytes against gastric cancer cell lines of lymphoid cell relate to the expression degree of the 4-1BBL in the gastric cancer cells. MKN-28 and SGC-7901 express high level of 4-1BBL, MGC-803 and BGC-823 express low level of 4-1BBL, The killing activity of lymphocytes against MKN-28 and SGC-7901 are higher than MGC-803 and BGC-823 gastric cancer cell lines.4 The gastric cancer cell 4-1BB expression is more high, IL-2,TNF-αthe level be more low, but IL-10 expressions be more high.The gastric cancer cell 4-1BBL expression is more high, IL-2, TNF-αthe level be more high, but IL-10’s expressions be more low.Hint the 4-1BB stimulates activate of path with the 4-1BBL to secrete with cell factor a certain relation.In a word, gastric cancer cell immunity relate to the expression of 4-1BB/4-1BBL, 4-1BBL the expression increases in the gastric cancer cell lines high strengthenned to kill wound function to the immunity of tumor cell, 4-1 BBL expression low, can’t effectively build up a 4-1BB/4-1BBL to lie to lead co- stimulate signal path, cause body immunity surveillance the function lower and immunity happen to escape.But the 4-1BB express in the gastric cancer cell lines and function is contrary to 4-1BBL, as for how to moderate the relation of them need a further research confirmation.

The Study on the Expressions of 4-1BB and 4-1BBL in Different Gastric Cancer Cell Lines

Abstract

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