臺灣博碩士論文加值系統

在本實驗室先前的實驗中,發現MDCK細胞培養於膠原蛋白膠質之中,會
先增殖形成細胞團,然後細胞團中間的細胞逐漸產生細胞凋亡的現象而使
之變成曩腔構造.為了證明是否阻止細胞凋亡就可避免曩腔狀構造的產生,
實驗室轉植了一個讓細胞較不易死的基因Bcl-2到MDCK細胞中,得到四個含
Bcl-2的細胞株B1,B4,B6,B9和一個plasmid control C1.我們發現過度表
現Bcl-2的細胞株被種於膠原蛋白膠中時主要會形成細胞團,表示細胞凋亡
是產生曩腔狀結構的一個重要步驟.另外,我們也發現B1和B9在膠原蛋白中
大約有25到30百分比的細胞團會產生類似管狀之結構,B6則幾忽完全不發
展成管狀結構.此外,本實驗室從MDCK選植出兩個次細胞株M411及M634.他
們培養於膠原蛋白中時會形成細胞團但不表現bcl-2,當這兩種細胞株種在
較低濃度的膠原蛋白中,它們會產生很劇烈的分支管狀結構.本論文的目地
主要想探討這些細胞株是如何產生管狀結構的機制.文獻中已知肝細胞生
長因子(HGF,Hepatocyte growth factor)可以藉由增加 urokinase-type
plasmin activator (uPA) 和uPA接受器之mRNA而促使MDCK產生管狀的結
構.因為uPA可以活化plasminogen變成plasmin,而 Plasmin又可以活化
Matrix Metalloproteinases(MMPs),所以活化型之MMPs可以藉由把細胞周
圍之基質(matrix)分解掉而讓細胞比較容易往外生長,這是一般血管新生
和腫瘤細胞漫延的常見基制.為了測試這幾個細胞株是否也經由活化uPA和
其下游調控的MMPs來達成長出管狀結構之目地,我測試了這些細胞株的uPA
和MMPs的表現.我發現B1和B9有比B4和B6還高的uPA活性表現,以Western
Blot來看,只有B6的uPA蛋白質表現較少,但是在不同濃度的膠原蛋白中時
uPA蛋白質的含量並不受影響而改變,另外,bcl-2的含量並不與uPA分泌量
成正向關係,這些結果顯示在MDCK細胞產生管狀結構的機制中uPA較bcl-2
佔有重要地位.另一方面,M411和M634細胞株卻沒有如B1和B9一樣高的活性
表現,uPA蛋白質含量也沒有特別多,而且以PAI-1(uPA之inhibitor)及6-
aminocaproic acid (plasmin 之inhibitor)無法完全抑制管狀結構之形
成,表示可能還有其它的因素影響管狀結構的形成.另外,我利用
GelatinSubstrate zymography 的方法來測量其MMPs之活性,可看出細胞
養於培養皿上時,MDCK細胞大致會產生92kD MMP (MMP9,又稱92kD
gelatinase)及72kD MMP (MMP2,又稱72kD gelatinase)兩種MMPs,當細胞
種於不同濃度的膠原蛋白中時,只有B6細胞株的MMP9表現較少,這結果顯示
MMP9的表現可能是MDCK細胞長出管狀結構的重要因素之一.

Previously, our laboratory discovered that MDCK cells cultured
in collagen gel formed cell aggregate and subsequently developed
central apoptosis that resulted in cyst cavitation. In order to
test whether prevention of apoptosis could avert cyst
cavitation, we transfected MDCK cells with bcl-2. Four bcl-2 and
one control-plasmid transfectants, namely B1, B4, B6, B9 and C1,
were obtained. We observed that all the bcl-2 transfectants
developed cell aggregates instead of cyst, indicating apoptosis
is an essential step for cyst formation. However, we also found
that about 25-30% of B1 and B9 clones developed tubule-like cell
aggregates in collagen gel but B6 did not.Moreover, we
identified two single clones from MDCK cells namely M411 and
M634 which developed mostly cell aggregates in collagen gel, but
did not express bcl-2. When these clones were seeded in collagen
gel of lower concentration, they developed branching tubule
vigorously. Activation of urokinase-type plasminogen activator(
uPA) and uPA receptor mRNA has been shown to be essential for
hepatocyte growth factor-induced MDCK branching tubulogenesis.
uPA convert plasminogen to the active form, plasmin, a serine
protease which activates a cascade of proteolytic event like the
activation of Matrix Metalloproteinases(MMPs). Active MMPs
degrade the matrix around the cells, promoting the cells extrude
and become tubule-like cell aggregates. This mechanism has been
shown to play a key role in tumor invasion and angiogenesis. To
delineate whether the activation of uPA and subsequent MMPs was
associated with the potential of tubulogenesis, we assessed both
urokinase(uPA) and MMP activity. We found that B1 and B9
contained higher uPA activity than B4 and B6. By uPA Western
blot, B6 has the least amount of uPA protein and there is no
difference between cells in different concentration collagen
gel. However, bcl-2 levels in these transfectants did not
parallel with their uPA activity. These results indicates that
uPA activity is more important than bcl-2 levels in tubulogensis
of MDCK cells. Nevertheless, uPA activities of M411 and M634
clones are not as high as those of B1 or B9. On the other hand,
PAI-1(plasminogen activator inhibitor) and 6-aminocaproic acid(
inhibitor of plaasmin) can not inhibit the tubulogenesis of B1
and M634 completely means that there are other factors involved
in tubulogenesis. Using Gelatin substrate gel zymography, we
showed that MDCK cells growth on dish express both 92kD MMPs(
MMP9) and 72kD MMP(MMP2). When the cells growth in different
concentration collagen gel, only B6 has less expression of MMP9.
The results of MMP expression correlates with the degree of
tubulogenesis well. These results indicate that the expression
of matrix metalloproteases could be an important factor for MDCK
cells tubular morphogenesis.