陰道鞭毛蟲是一種具有鞭毛的寄生性原蟲，主要侵犯生殖泌尿道鱗狀上皮細胞。全球每年約有1.56億人口被侵犯。儘管許多患者無症狀，但感染陰道鞭毛蟲可能會導致人類鞭毛蟲症、生殖泌尿道急慢性病變，甚至生殖泌尿細胞癌化。陰道鞭毛蟲臨床蟲株間，黏附能力、細胞毒性差異甚大；特別是American Type Culture Collection (ATCC) 50167，具有高黏附能力及細胞毒性。既然子宮頸上皮細胞是陰道鞭毛蟲宿主傷害的主要標的，本研究從高致病力陰道鞭毛蟲ATCC 50167著手，試圖從分析比較陰道鞭毛蟲不同分離株差異，進一步了解陰道鞭毛蟲的子宮頸上皮細胞傷害活性。實驗模式的子宮頸上皮細胞，選用癌化細胞株，人類乳突病毒核酸陽性子宮頸癌上皮細胞株HeLa。除了ATCC 50167為對照依據外，陰道鞭毛蟲另選用五株不同臨床分離株，包括ATCC 30001、ATCC 30236、ATCC 30238、ATCC 50142、ATCC 50148，藉以比較不同陰道鞭毛蟲臨床分離株的蟲體特性差異。生長曲線實驗結果發現，從培養後0小時到培養後24小時間，不同株陰道鞭毛蟲生長數量持續上升，與ATCC 50167比較，ATCC 30001、ATCC 50148生長滿度較大；ATCC 50142生長滿度較小；ATCC 30236、ATCC 30238與ATCC 50167相近；這六株陰道鞭毛蟲的生長指數中期，都是培養後約16小時。黏附是已知最主要的致病機制，而陰道鞭毛蟲彼此聚集越多，黏附能力通常也越強，因此，實驗透過觀察蟲體聚集，確立不同蟲株毒性；接種在培養盤1小時後，陰道鞭毛蟲以自由游動、獨立蟲體型態為主。實驗發現，ATCC 50167陰道鞭毛蟲本身具有較高的蟲體聚集性，聚集蟲體群落型態最為顯著。陰道鞭毛蟲在HeLa細胞黏附方面，ATCC 50167及ATCC 50142對於細胞具有較高的黏附性；ATCC 50148黏附最少。細胞剝離及漂浮是陰道鞭毛蟲共培養後最顯著的細胞病變，ATCC 50167的HeLa細胞傷害最強；ATCC 30001、ATCC 30236、ATCC 30238居中；ATCC 50142、ATCC 50148的細胞傷害最弱。陰道鞭毛蟲的細胞傷害作用與陰道鞭毛蟲黏附蛋白、細胞連接蛋白、細胞激素有關。陰道鞭毛蟲與HeLa細胞共培養後，AP33、AP65、AP120，三種陰道鞭毛蟲黏附蛋白mRNA變化方面，ATCC 30001降低AP33/AP65；ATCC 30236降低AP33，增加AP65/AP120；ATCC 30238增加AP33/AP120；ATCC 50142增加AP65/AP120；ATCC 50148降低AP65/AP120；ATCC 50167降低AP33/AP65，其它變化不顯著。ATCC 50167會降低細胞連接蛋白Connexin-43蛋白質表現量。ATCC 30001、ATCC 30236、ATCC 30238、ATCC 50142、ATCC 50148、ATCC 50167都顯著增加interleukin-6 (IL-6) mRNA表現；只觀察到ATCC 30236、ATCC 30238、ATCC 50142、ATCC 50148增加tumor necrosis factor alpha (TNF-α) mRNA表現；ATCC 50148增加intercellular adhesion molecule-1 (ICAM-1) mRNA表現。
總合實驗結果，陰道鞭毛蟲分離株間，具有生長特性差異、聚集型態差異、細胞黏著貼附差異，陰道鞭毛蟲蟲體聚集能力，與HeLa細胞黏附能力結果一致，但在細胞傷害，只有部份蟲隻顯現對應相關性，像是ATCC 50167。但是，黏附力與黏附蛋白表現量、細胞傷害、細胞連接蛋白表現量、細胞激素表現量，卻沒有絕對一致性。結果說明，陰道鞭毛蟲黏附力，並不是細胞毒性、蟲體細胞互動作用的唯一決定性因素，陰道鞭毛蟲也有可能透過一些接觸非依賴性 (contact independent) 的方式去影響宿主細胞。本實驗觀察發現陰道鞭毛蟲特性差異，希望實驗發現能有助於陰道鞭毛蟲與子宮頸上皮細胞傷害關聯性的研究。

Trichomonas vaginalis (T. vaginalis) is a flagellated parasitic protozoan, with an estimated 156 million people infected globally each year. Genitourinary squamous epithelial cells are the primary targets of T. vaginalis. Although many infected patients remain asymptomatic, infection of T. vaginalis is of high risk to develop clinical trichomoniasis and a range of complications, including acute and chronic genitourinary diseases and even carcinogenesis. Clinical T. vaginalis isolates have distinct adhesive and cytotoxic properties. ATCC 50167 is a strain of T. vaginalis isolate characterized by high adhesive and cytotoxic properties. Cervical epithelial cells are vulnerable to the infection of T. vaginalis. To reveal potential relationship between T. vaginalis and cervical epithelial cell injury, T. vaginalis clinical isolates were first analyzed and compared by characterizing growth curve, morphology, and cellular actions on cervical epithelial cells, compared with high pathogenic ATCC 50167. Therefore, our study is aiming to explore the biological properties of T. vaginalis clinical isolates towards cervical epithelial cell injury. Experimental cell models of cervical epithelial cells were created by using cancerous cell line: HPV-positive HeLa cells. In addition to ATCC 50167, other five clinical T. vaginalis isolates with distinct phenotypes were included to investigate and compare phenotypic variations and cellular actions on cervical epithelial cells. The used T. vaginalis isolates were ATCC 30001, ATCC 30236, ATCC 30238, ATCC 50142, and ATCC 50148. Phenotypic variations of each T. vaginalis isolate were first characterized and compared centered on growth dynamics and aggregative properties. Data of growth curve study indicated that when compared with ATCC 50167, the ATCC 30001 and ATCC 50148 had the highest growth density and the ATCC 50142 was the least. All the T. vaginalis isolates reached mid-log phase 16 hours after incubation. Adhesion is the important pathogenic determinant, and the more the parasite aggregate to one another, the more adherence. Thus, study was designed to measure aggregative property of T. vaginalis isolates for the evaluation of virulence. One hour after plating onto culture plates, most T. vaginalis were single with rounded morphology. Among the T. vaginalis isolates, aggregate morphology was apparent in ATCC 50167. In evaluating adhesive properties of T. vaginalis isolates on HeLa cells, ATCC 50142 and ATCC 50167 had the highest adhesive properties and the ATCC 50148 was the least. Cell detachment and floating were remarkable cytopathic change after T. vaginalis infection. ATCC 50167 showed the most severe cytopathic change on HeLa cells and followed by ATCC 30001/ATCC 30236/ATCC 30238 and then ATCC 50142/ATCC 50148. Adhesion proteins of T. vaginalis, cell junction proteins, and cytokines have been implicated in the cytotoxic effects of T. vaginalis. Co-culture of HeLa cells with T. vaginalis, contents of AP33, AP65, and AP120 mRNA varied. ATCC 30001 decreased AP33/AP65 expression; ATCC 30236 decreased AP33 but increased AP65/AP120 expression; ATCC 30238 increased AP33/AP120 expression; ATCC 50142 increased AP65/AP120expression; ATCC 50148 decreased AP65/AP120 expression; ATCC 50167 decreased AP33/AP65 expression. Other expressions were not significantly differed. Reduction of cell junction Connexin 43 protein was only seen in ATCC 50167. ATCC 30001, ATCC 30236, ATCC 30238, ATCC 50142, ATCC 50148, and ATCC 50167 all increased expression of IL-6 mRNA. Elevated expression of TNF-α mRNA was observed in ATCC 30236, ATCC 30238, ATCC 50142, and ATCC 50148. Only ATCC 50148 increased expression of ICAM-1 mRNA.
In summary, T. vaginalis clinical isolates had phenotypic variations and cellular actions on cervical epithelial cells, including growth dynamics, morphology, adhesive properties, cytopathic change, T. vaginalis adhesive proteins, cellular junction proteins, and cytokine expression. The ability of T. vaginalis to aggregate was consistent with their adhesion to HeLa cells; however, regarding cytopathic effects, only some strains - such as ATCC 50167 - showed a corresponding correlation. Adhesive ability of T. vaginalis was not correlated well with adhesion protein expression, cytotoxic effects, cell junction protein expression, and cytokine expression. These results indicate that the adhesive capability of T. vaginalis is not the sole determinant of cytotoxicity or parasite-host cell interactions. T. vaginalis may also affect host cells via contact-independent mechanisms. Data of our findings suggest that T. vaginalis isolates have phenotypic variations and unique biological implications. We hope our findings of T. vaginalis phenotypic variations and cellular actions on cervical epithelial cells may provide basis to advance the study of relationship between T. vaginalis and cervical epithelial cell injury.

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