太平洋紫衫醇是治療癌症最成功的藥物之一，它治療的功能以穩定tubulin為標的使得細胞分裂無法繼續進行而導致細胞凋亡，雖然太平洋紫衫醇是很成功的抗癌藥物，然而肺癌治療所產生的抗藥性使得肺癌治癒率偏低約為10~15%，抗藥性儼然已經成為臨床用藥的最大阻礙。
太平洋紫衫醇用於惡性腫瘤的治療目前正積極研究中，在非小細胞及小細胞肺癌，頭頸部鱗狀上皮癌，膀胱癌，生殖細胞瘤等均報告有相當療效，文獻顯示，單獨使用太平洋紫衫醇250mg/m2於先前沒有治療過的小細胞肺癌其療效可達34%，對先前沒有治療過的非小細胞肺癌也有21%至24%的療效。
我們利用太平洋紫衫醇培養抗性細胞株(A549R)並比較A549細胞株與A549R抗性細胞株的差異蛋白，再利用MTT assay來測試太平洋紫衫醇在不同濃度下對細胞株生長的影響，以及太平洋紫衫醇治療的肺癌病患檢體，透過二維電泳，來分析出多種蛋白質差異性的表現，比對正常細胞、無抗性及有抗性細胞的表現與文獻所研究之抗藥性路徑的關係性。
本實驗在培養抗性細胞時發現太平洋紫杉醇和鈣離子會調控細胞株生長，由於鈣離子的調控因而影響蛋白質的改變而產生抗性。利用二維電泳 patterns比對出A549細胞株與A549R抗性細胞株以及肺癌病患檢體正常細胞與抗性腫瘤細胞的蛋白質表現時發現太平洋紫衫醇與鈣離子相互作用是產生抗性的主要因素，從肺癌臨床治療統計得到驗證；許多惡性腫瘤都會產生高血鈣現象，而高血鈣的病患其腫瘤的惡性程度比較嚴重，由上述可得知鈣離子的確是產生抗藥性的主要因素。

Paclitaxel is one of the most successful drugs for the treatment of cancer because of its ability to target tubulin, block cell cycle progression at mitosis, and induce apoptosis. Despite the success of Paclitaxel, the development of drug resistance hampers its clinical applicability. Paclitaxel is used in malignant tumors in the present research, including non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), head-neck scale epitheliomatous, urinary bladder cancer, tumor of the reproduction organ and so on. Clinical treatment of paclitaxel be injected 250mg/m2 to previously non-treated patients of small lung cancer its effect about 34% and previously non-treated patients of non-small lung cancer its effect about 21% to 24% .
On the other hand we had established a taxol-resistant human lung carcinoma subline A549R by paclitaxel to compare the different proteins with A549 wild type and treat the different concention of paclitaxel with MTT assay so as to observe the tolerance dosage of subline growth.We obtained the patient’s specimens of lung cancer to treat with paclitaxel some of resistant and some of non-resistant to compare differentially expressed proteins between normal and tumor. When we cultured taxol-resistant human lung carcinoma subline in which paclitaxel and calcium regulate growth, owing to the proteins of changes result to resistance. The extraction cell subline and specimens were analyzed by 2-D electrophoresis patterns and we found that interact paclitaxel with calcium it is the important factor of drug resistant. Verified from clinical treatment might have hypercalcemia in malignant tumors and calcium ion may increase paclitaxel drug resistance and hypercalcemia patient will be more insensitive to paclitaxel treatment.

1Introduction........................................................................1
1.1Background…………………............................................1
1.1.1 Lung Cancer…………………………………………......2
1.1.2Paclitaxel……………………………………………….....4
1.1.3 Multidrug-resistant……………………………………...5`
1.1.4 Biomarkers……………………………………………....7
1.1.5 Similar Performed Studies…………………………….7
2. Experiment flowchart……………………………………..11
2.1 Study flowchart…………………………………………11
2.2 Experiment flowchart……………………………………11
2.3 A549 and A549R cell line protein extraction…………12
2.4 Specimens normal and tumor protein extraction……12
2.5 Identified and analyzed differential proteins…………13
2.6 MTT asssy test A549 cell line of paclitaxel drug resistant............................................................................13
3. Materials and Methods……………………………………14
3.1 Cell Line Culture…………………………………………14
3.1.1 A549 Cell Line Culture.............................................14
3.1.2 A549-R Cell Line Culture……………………………17
3.1.3 The Specimens Are Gifted the Lung Cancer Patients Tissues from E-Da Hospital. ……………………………..18
3.2 Protein Extraction.....................................................19
3.2.1 A549 Cell Line Protein Extraction.......................19
3.2.2 Tissue Samples Protein Extraction........................19
3.3 Two-dimensional Electrophoresis (2-DE)...................20
3.3.1 Run IEF..................................................................22
3.3.2 Run 2-DE...............................................................25
3.3.3. Run Two-dimensional Electrophoresis Patterns.....28
3.4 MTT............................................................................28
3.5 In Gel Digestion.........................................................29
3.6 TOF-MS.....................................................................30
4. Result…………………………………………………….32
4.1 Study Raises Concerns about Using A549 Cell to Test Drug Sensitivity and Drug-resistance………………………32
4.1.1 Calcium and Cholesterol Increase Chemoresistance of Cells…………………....................................................32
4.1.2 Two-dimensional Electrophoresis Analysis of Tissue, A549 and A549R Cells Line...............................................32
4.1.3 Multivariate Statistics Analysis……………...............33
5. Discussion………………………………………………....36
5.1 Mechanisms of Drug Resistantance of Discussing…..36
5.1.1 Drug Resistant of Molecular Mechanisms………….36
5.1.2 Drug Resistant Biochemical Mechanisms………….36
5.1.3 The Study Hypothetical Model of Discussing………36
5.2 Establishment of Paclitaxel Resistant Human Lung Cancer Cell Line of Discussing…………………………….37
5.3 Drug Resistance Simply Tests for Discussing……….37
5.4 The Difference Proteins Played in the Biological Process Being Introduced and Discussed…………………38
5.5 Drug Resistance and Calcium Interact in Human Lung Cancer of Discussing.........................................................38
6. Conclusion………………………………………………....40
7. Future work....................................................................41
7.1 A Data Survey of Lung Cancer Clinical Treatments…41
7.1.1 The Project Combines with Lung Cancer Treatment Relations............................................................................41
7.1.2 The Concrete Achievement…………………………..42
7.1.3 Definition of Statistical Factor Models………………42
8. References...................................................................43
9. Figures and Tables Countents……………………….....52
Future 1-1 Paclitaxel structure……………………………....5
Future 1-2 Summary of the observed effects of submicromolar paclitaxel.....................................................6
Figure 3-1 A549 cell line growth curve………....................16
Figure 3-2The statistical analysis of the standard curve…18
Figure 3-3 pipette the appropriate volume each strip holder.................................................................................23
Figure 3-4 Amersham Pharmacia Biotech IPGphor IEF…24
Figure 3-5 SE 600 Ruby…………………………………….26
Figure 3-6 Typhoon 2D gel scanner……………………….28
Figure 4-1 A549 cell line in FBS free medium…………….54
Figure 4-2 the drugs paclitaxel and carboplatin original fluid’s concentrations……..................................................55
Figure 4-3 the drugs paclitaxel and carboplatin original fluid’s concentrations……..................................................55
Figure 4-4 We compared with two-dimensional electrophoresis patterns cell lines….................................56
Figure 4-5 osmotics pressure be changed to D3 and D5 in the cell membranes……...................................................57
Figure 4-6 transcriptions be changed to D17 and D18 in the nuclear……….............................................................57
Figure 4-7 translations be changed to D9 and D14 in the cytoplasm………...............................................................58
Figure 4-8 the infections was detected in the specimens T6 and T7………………….....................................................58
Figure 4-9 osmotics pressure vs ros in the specimens T16 and T17…………………...................................................59
Figure 5-1 the hypothetical model…………………………………………………............59
Figure 7-1 the hypothetical model of survey of lung cancer clinical treatment…….........................................................60
Table 3-1 General example using RPMI 1640 media........14
Table 3-2 Culture cell growth…………………………….....16
Table 3-3 Paclitaxel drug concentration.............................17
Table 3-6 protein concentration measurement..................18
Table 3-7 to prepare the rehydration buffer…………….....23
Table 3-8 IEF procedure design……………………...........24
Table 3-9 Strip equilibrations………………………………..24
Table 3-10 Casting gel protocol…………………………….25
Table 3-11 Fixation solution…………………………………26
Table 3-12 Stain and destain solution……………………...27
Table 3-13 Silver stain and step…………………………....27
Table 3-14 MTT Basic protocol……………………………..29
Table 4-1 the proteins of reaction related calcium up regulation...........................................................................34
Table 5-1 the patterns were analyzed the changes of the proteins……………….........................................................38
Table 3-4 Paclitaxel drug resistant (A549-R) cell line culture................................................................................52
Table 3-5 The lung cancer specimens…………….............53
10. Appendix………...........................................................61

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