Expression And Clinical Significance Of Survivin Gene In Non-small Cell Lung Carcinoma

Lung cancer is one of the common malignant tumors and the incidence of the disease rises in recent years. At present, the diagnosis and treatment of lung cancer are progressed continuously, but the mortality rate has no obvious drop. Lung cancer has become the serious malignant tumor, which menaces human s health.Nowadays, in peoples opinion, the incidence of lung cancer is not only the abnormity of differentiation and proliferation, but also the abnormity of apoptosis. Apoptosis is the most important physiological procedure of controlling cell natural metabolize. The abnormity of apoptosis will cause over - proliferation and disorder growth of cells. Thus, the research on the concerning factors of tumor will assist the study of prevention and cure of the disease. It will also be helpful in adopting correspondent measures, which will improve the effect of tumor treatment when dealing with the apoptosis misbalance of tumor. Recently, the research on molecule mechanism of apoptosis is developing fast. And until now, a few proteins which involve in the apoptosis procedure have been found out, including such as Fas and FasL system, ICE family, bcl - 2 protein family, p53, IAP family and adeno virus El A, E1B and so on.Inhibitor of apoptosis protein (IAP) is a key factor, which restrains apoptosis. The function of inhibitor of apoptosis protein family is far more active than the function of bcl - 2 family. However Survivin is a newly discovered member in IAP family, which is the smallest IAP protein known by now. But it is maybethe mighty inhibitor of apoptosis gene. The gene is 15 kb in total length and locates 17p25. Survivin gene possesses four exon and three intrinsic factor . It has an open reading frame in 2811 and 2560 - 2920 is the non - TATA initiation factor and GC enrichment area of Survivin gene. Survivin gene in tumor expresses selectively. It has been discovered that the expression of Survivin exists in the tumor multi -metastasis cells. On the contrary, it will express none or less in the normal mature tissues. Experiments have verified that Survivin gene prevents the procedure of apoptosis directly, mainly through inhibiting the activity of Caspases -3,7. It can also combine with periodic and adjustable gene CDK4, to form a mixture to inhibit the activity of Caspases - 3, which resists apoptosis indirectly. These will lead to the over - proliferation of cells, even cause the incidence of tumor.Just because Survivin gene has the mighty function in inhibition of apoptosis, it plays an important role in the generant and developing stage. So, detecting the expression of apoptosis in tumor tissue has significant effect in the clinic practice. In the past, the research focusing on Survivin gene in the expression of multi - tumor tissues, but the expatiation of the expression in lung cancer tissue is rare. Therefore, our experiment probes the expression of Survivin gene in lung cancer through chemical methods of immune tissue. Further more, it studies the inner relations with the expression of Survivin gene, tissue types, clinic stages, lymph metastasis and so on. Then, the research provides the theoretical base for lung cancer diagnosis and the judgment of state of illness. Then effective treatment will be taken according to the diagnosis and judgment.Material and Methods1. Clinical materialsCollecting 50 cases, which are primary non — small cell lung cancer tissues, all the cases are not treated with radiotheraphy and chemotherapy. Male 35 cases, female 15 cases; age 40 -79, average age 52. 3. Squamous carcinoma 25 cases, adeno - carcinoma 21 cases, adeno - squamous carcinoma 4 cases; TNM stages; T1 4 case, T2 30 case, T3 13 case, T4 3 case. Degrees ofdifferentiation; high, middle differentiation 40 cases, low differentiation 6 cases , adeno - squamous carcinoma 4 cases, lymph metastasis 27 cases, non -lymph metastasis 23 cases.2. Main reagent and instrumentThe specimen of lung cancer is fixed with formalin, buried by paraffin, sliced continuously, tissue thick 5 m. Immune tissue chemical chromosome a-dopts SP method. At the same time, preparing normal lung tissues which is 5 cm from the tumor and make a contrast. Survivin multi - clone antibody with the type rabbit, SP prompt reagent box type, is supplied by Fujian Mai Xin Bioengi-neering Co, Ltd. . The steps of manipulation are conducted according to manual instruction strictly.3. MethodAll the tissues from lung cancer patients are dealt with immune tissue chemical dye, adopting SP method. According to pathology types, degrees of differentiation, TNM stages, lymph gland shift or not, tissues are divided into groups and made statistics analysis.[1] Adopting of the tissue materials. Bury 50 specimens of lung cancer with Paraffin wax and slide each wax, tissue thick 5 m - B, and conduct immune dye.[2] Deparaffinization with slides and dewaxes with slides 15 minutes.[3] Hydration in the gradient alcohol.[4] Distilled water washed the slides for 5 minutes, PBS washed the slides 5 minutes for 3 times.[5] Deactivation of endogenous peroxides. Put in the 3% H₂O₂ PBS (135mlPBS +30% H₂O₂5ml) with in room temperature for 5 minutes.[6] PBS washed the slides 5 minutes for 3 times.[7] Put slides into EDTA antigen restore fluid for 10 minutes.[ 8 ] PBS washed the slides 5 minutes for 3 times.[9] Add immune goat serum in the box immune serum for 15 minutes.[10] Add 1 ;100 diluted Survivin first antibody, overnight at 4 centigrade, in room temperature for 30 minutes. PBS washed the slides 5 minutes for 3 times.[11] Add second antibody marked with organist, protect and hatch for 30 minutes under 37 centigrade in the box. PBS washed the slides 5 minutes for 3 times.[12] Dripped the SP and hatch for 30 minutes under 37 centigrade in the box. PBS washed the slides 5 minutes for 3 times.[13] DAB (3,3 -diaminobenzidine) develops the color. When the suitable result appear under microscope, wash the slides in running water to end dye.[14] Dyed for one minute to reply, wash the slides in running water for 10 minutes. Dehydrated, transparent and sealed slide.Control: the tissue section of the known digestive duct serves as positive control, the PBS is used in place of the first antibody and serves as blank control, and the surrounding normal tissue serve as normal control.4. Immune Group Dye result judgeThe dyed cell liquid, which is pale brown, is positive. Picking up 5 randomly under high power visions of observation ( magnify 400 times) , counting positive cell percentage. The percentage of positive cell > 10% is the positive result.5. Analysis statisticsDealing with SPSSIO. 0 software, the diversity of positive rate in different groups could be checked with \ .Result1. Expression of Survivin gene in lung cancer and normal lung tissue In all 50 cases of lung cancer specimens, the positive expression of Survivin gene are 39 cases, negative expression are 11 cases, 78.0% positive rate in total. But in the normal lung tissue the positive expression of Survivin are only 3 cases, negative expression are 47 cases, expression rate is 6. 0%. The difference of the two positive expressions is obvious ( p <0.05). The expression of Survivin gene mainly locates in cell liquid, and they look yellow or pale brown.