(Comparison of Six Cadherin Subtypes in Endometriosis and Eutopic Endometrium)
3-1： 材料與方法
2-1-1： 檢體與取樣(tissues)
本實驗檢體取自在中國醫藥學院附設醫院婦產科接受子宮內膜異 位囊腫切除手術所之檢體，患者年齡為 20~42 歲的生殖年齡(reproductive age)，開刀前三個月未曾使用過賀爾蒙藥物。取出之檢體剝取內膜囊腫的 裡層子宮內膜異位組織，而正常子宮內膜的組織則以刮匙刮取少許子宮腔 內之子宮內膜，部份以液態氮急速冷凍隨即保存於-80°C 冰箱中，另一部份 以 4% formaldehyde/2% glutaraldehyde 固定，以 hematoxylin & eosin 染 色，確定子宮內膜的組織是處於增殖期或分泌期，其中增殖期 5 位，分泌 期 4 位。
3-1-2：RNA 之萃取(Trizol RNA extraction)：
其所需儀器、藥劑及步驟如前所述(見第 22 頁) RNA 產物之定量
â 2
µ
l RNA +3µ
l ddH2O + 5µ
l RNA denature buffer â 加熱 58°C15 分鐘â 置於冰上 2 分鐘 â 短暫離心
â 加入 2
µ
l RNA loading buffer â 跑膠â 置於 EBTBE 內染色 30 分鐘 â 置於 UV box 照相
â 評估 RNA 相對含量。
3-1-3： RT(Reverse transcription, Promega) 其所需儀器、藥劑及步驟如前所述(見第 24 頁)
3-1-4：PCR
其所需儀器、藥劑及步驟如前所述(見第 25 頁)
â 首先以 PCR 的方式得到的 GAPDH(a house keeping gene)，再以 不同的 PCR cycle 求得最適當的 cycles 為 25 cycles
â 再以相同的 PCR 的方式求得 E-cad、P-cad、N-cad、cad-6、cad-9、
cad-11，最適當的 cycles。
â 結果以不同的 PCR cycle 求得最適當的 cycles 分別為 GAPDH 25 cycles，E-cad、P-cad、N-cad、cad-6、cad-9、cad-11 為 35 cycles (見 Figure 6-9)
â 算出子宮內膜異位細胞與子宮腔內之子宮內膜細胞之間所需的 mRNA 量
â 以六種不同鈣粘蛋白亞型的specific cadherin primers 製造鈣粘蛋白 產物(cadherin products)
Figure 6. Validation of semiquantitative RT-PCR for GAPDH or E-cad.
Total RNA isolated from eutopic endometrium (A) and endometriotic lesion (B) were reverse transcribed. An aliquot of first strand cDNA was amplified for GAPDH or E-cad using different numbers of PCR cycles.
A linear relationship was observed between PCR products and amplification cycle when plotted with other two independent experiments (data not shown)
Figure 7. Validation of semiquantitative RT-PCR for P-cad or N-cad. Total RNA isolated from eutopic endometrium (A) and endometriotic lesion (B) were reverse transcribed. An aliquot of first strand cDNA was amplified for P-cad or N-cad using different numbers of PCR cycles. A linear relationship was observed between PCR products and amplification cycle when plotted with other two independent experiments (data not shown)
Figure 8. Validation of semiquantitative RT-PCR for cad-6 or cad-9. Total RNA isolated from eutopic endometrium (A) and endometriotic lesion (B) were reverse transcribed. An aliquot of first strand cDNA was amplified for cad-11 using different numbers of PCR cycles. A linear relationship was observed between PCR products and amplification cycle when plotted with other two independent experiments (data not shown)
Figure 9. Validation of semiquantitative RT-PCR for cad-11. Total RNA isolated from eutopic endometrium (A) and endometriotic lesion (B) were reverse transcribed. An aliquot of first strand cDNA was amplified for cad-11 using different numbers of PCR cycles. A linear relationship was observed between PCR products and amplification cycle when plotted with other two independent experiments (data not shown)
3-1-5：以半定量 RT-PCR 分析內膜異位組織和子宮內膜組織中六種鈣黏蛋 白亞型的不同表現
â 將 所 得 到 的 鈣 粘 蛋 白 產 物 (cadherin products)相 片 以 Hewlett Packer Digital scanner 掃瞄至電腦分析之
â 使用 UN-SCAN-IT Gel Version 5.1 system (Silk Scientific, Orm, Utah)分析求得 Cadherin/GAPDH mRNA 之比值 。 ( Figure 10 &
11)
3-1-6：統計方法
以 Wilcoxon signed ranks test 來作檢定，以 p<0.05 為有顯著意義。
3-2： 結果
第二階段實驗我們發現這六種鈣粘蛋白同樣也存在子宮腔內之 子宮內膜組織中。其中 N-cad 和 Cad-9 文獻上尚未有人報告過存在於子宮 內膜，屬於新的發現。還有，E-cad、N-cad、Cad-6、Cad-9 及 Cad-11 的 表現，在子宮內膜異位組織與子宮腔內之子宮內膜之間並無統計學上的差 異。唯有 P-cad 在子宮內膜異位組織的表現較較子宮腔內之子宮內膜高，
呈統計學上有意義的差別，這樣的差異無論是在增生期或分泌期都是存在 的(見 Figure 10 & 11)。
Figure 10. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the proliferative phase of the menstrual cycle. Photomicrographs of ethidium bromide-stained gels containing PCR products generated using primers for E-cad P-cad or N-cad at 361, 357 and 373 base pairs respectively (Panel A) or cad-6, cad-9, or cad-11 at 275, 416 and 742 base pairs respectively (panel B).
For each cadherin subtype, PCR products were generated using template cDNAs generated from eutopic endometrium (lane a) or endometriotic lesions (lane b). A PCR reaction in which the first strand cDNA was omitted was performed as a negative control (lane c).
Products amplified using GAPDH specific primers at 359 base pairs are also shown for each sample. DNA markers are presented on the left hand-side of each gel.
The absorbance values obtained for each cadherin subtype were normalized to the values obtained from for the corresponding GAPDH.
The results derived from this analysis as well as from two other independent experiments (photomicrographs not shown) were standardized to the control. The values obtained from this study, as well as from 4 other studies are presented (mean ±SEM; n=5) in the bar graphs. (*p<0.05).
Figure 10. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the proliferative phase of the menstrual cycle.
Figure 11. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the secretary phase of the menstrual cycle. Photomicrographs of ethidium bromide-stained gels containing PCR products generated using primers for E-cad P-cad or N-cad at 361, 357 and 373 base pairs respectively (Panel A) or cad-6, cad-9, or cad-11 at 275, 416 and 742 base pairs respectively (panel B).
For each cadherin subtype, PCR products were generated using template cDNAs generated from eutopic endometrium (lane a) or endometriotic lesions (lane b). A PCR reaction in which the first strand cDNA was omitted was performed as a negative control (lane c).
Products amplified using GAPDH specific primers at 359 base pairs are also shown for each sample. DNA markers are presented on the left hand-side of each gel.
The absorbence values obtained for each cadherin subtype were normalized to the values obtained from for the corresponding GAPDH.
The results derived from this analysis as well as from two other independent experiments (photomicrographs not shown) were standardized to the control. The values obtained from this study, as well as from 4 other studies are presented (mean ±SEM; n=4) in the bar graphs. (*p<0.05).
Figure 11. Comparison of the cadherin mRNA levels present in eutopic endometrium or endometriosis obtained during the secretary phase of the menstrual cycle.