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‫ﺗﺎرﻳﺦ درﻳﺎﻓﺖ‪1396/2/8 :‬‬ ‫ﻣﺠﻠﻪ داﻧﺸﻜﺪه ﭘﺰﺷﻜﻲ اﺻﻔﻬﺎن‬
‫ﺗﺎرﻳﺦ ﭘﺬﻳﺮش‪1396/3/27 :‬‬ ‫ﺳﺎل ﺳﻲ و ﭘﻨﺠﻢ‪/‬ﺷﻤﺎرهي ‪/430‬ﻫﻔﺘﻪي دوم ﺗﻴﺮ ﻣﺎه ‪1396‬‬

‫ﺑﻬﻴﻨﻪﺳﺎزي ﺗﺨﻠﻴﺺ اﺳﭙﻮراﻣﻴﻦ ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ ]‪ [Ipomoea Batatas (L.) Lam‬و ﺑﺮرﺳﻲ اﺛﺮ ﺿﺪ ﺗﻜﺜﻴﺮي‬
‫آن ﺑﺮ ﺳﻠﻮلﻫﺎي ﺳﺮﻃﺎن ﭘﺴﺘﺎن‪ ،‬ردهي ‪MCF-7‬‬

‫‪4‬‬
‫ﻣﺮﺿﻴﻪ ﻗﻴﻮﻣﻴﺎن‪ ،1‬ﻋﺎدﻟﻪ ﻋﻠﻲ ﻫﺎﺷﻤﻲ‪ ،1‬ﻣﺮﻳﻢ اﻣﻴﺪي اﺳﻜﻮﻳﻲ‪ ،2‬اﻳﺮج ﻧﻴﻜﻮﻛﺎر‪ ،3‬آزاده ﻛﺒﻴﺮي‬

‫ﻣﻘﺎﻟﻪ ﭘﮋوﻫﺸﻲ‬
‫ﭼﻜﻴﺪه‬

‫ﻣﻘﺪﻣﻪ‪ :‬از ﮔﺬﺷﺘﻪ ﺗﺎ ﺑﻪ اﻣﺮوز‪ ،‬ﻳﺎﻓﺘﻦ درﻣﺎنﻫﺎﻳﻲ ﺑﺎ ﻣﻨﺸﺄ ﮔﻴﺎﻫﻲ و روش اﺳﺘﺨﺮاج راﺣﺖ و ارزان ﺑﺮاي ﺑﻴﻤﺎريﻫﺎﻳﻲ ﻧﻈﻴﺮ ﺳﺮﻃﺎن ﻣﻮرد ﺗﻮﺟﻪ ﺑﻮده اﺳﺖ‪ .‬ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﻳﻦ ﻛﻪ‬
‫ﭘﺮوﺗﺌﺎزﻫﺎ در روﻧﺪ اﻳﺠﺎد و ﮔﺴﺘﺮش ﺳﻠﻮلﻫﺎي ﺳﺮﻃﺎﻧﻲ دﺧﻴﻞ ﻫﺴﺘﻨﺪ‪ ،‬ﻣﻬﺎر ﻛﻨﻨﺪﮔﺎن ﭘﺮوﺗﺌﺎزي را ﻣﻲﺗﻮان ﺑﻪ ﻋﻨﻮان ﮔﺰﻳﻨﻪاي ﺑﺮاي درﻣﺎن در ﻧﻈﺮ ﮔﺮﻓﺖ‪ .‬اﺳﭙﻮراﻣﻴﻦ‪ ،‬از‬
‫ﺟﻤﻠﻪ ﻣﻬﺎر ﻛﻨﻨﺪﮔﺎن ﺗﺮﻳﭙﺴﻴﻨﻲ اﺳﺖ ﻛﻪ در رﻳﺸﻪي ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ وﺟﻮد دارد‪ .‬در اﻳﻦ ﻣﻄﺎﻟﻌﻪ‪ ،‬روش اﺳﺘﺨﺮاج اﺳﭙﻮراﻣﻴﻦ ﺑﻬﻴﻨﻪ ﺷﺪ و ﺑﺮاي اوﻟﻴﻦ ﺑﺎر‪ ،‬اﺛﺮ آن ﺑﺮ ﺗﻜﺜﻴﺮ‬
‫ﺳﻠﻮلﻫﺎي ﺳﺮﻃﺎن ﭘﺴﺘﺎن ردهي ‪ (MCF-7) Michigan cancer foundation-7‬ﺑﺮرﺳﻲ ﮔﺮدﻳﺪ‪.‬‬

‫روشﻫﺎ‪ :‬ﻋﺼﺎرهي آﺑﻲ از ﺳﻴﺐزﻣﻴﻨﻲﻫﺎي ﺷﻴﺮﻳﻦ اﺳﺘﺨﺮاج ﺷﺪه‪ ،‬ﻣﺤﻠﻮل ﺷﻔﺎف ﺷﺪه وارد ﺳﺘﻮن ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ‪Diethylaminoethanol-Sepharose‬‬
‫)‪ (DEAE-Sepharose‬ﮔﺮدﻳﺪ‪ .‬ﭘﺲ از ﺷﺴﺘﺸﻮ ﺑﺎ ﺷﻴﺐ ﻧﻤﻜﻲ ﭘﻴﻮﺳﺘﻪ‪ ،‬ﺑﺨﺶﻫﺎي ﺧﺎﻟﺺ ﺷﺪه ﺟﺪاﺳﺎزي ﺷﺪﻧﺪ‪ .‬ﺑﺮاي ﺗﺄﻳﻴﺪ ﺧﻠﻮص ﭘﺮوﺗﺌﻴﻦ اﺳﺘﺨﺮاج ﺷﺪه از روش‬
‫‪ (SDS-PAGE) Sodium dodecyl sulfate polyacrylamide gel electrophoresis‬اﺳﺘﻔﺎده ﮔﺮدﻳﺪ‪ .‬ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎري اﺳﭙﻮراﻣﻴﻦ ﺧﺎﻟﺺ ﺷﺪه‪ ،‬ﺑﺎ اﺳﺘﻔﺎده از‬
‫روش ‪ Laskowaski-Takanara‬اﻧﺪازهﮔﻴﺮي ﮔﺮدﻳﺪ و در اﻧﺘﻬﺎ از روش ‪،(MTT) 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide‬‬
‫ﺑﺮاي ﺑﺮرﺳﻲ اﺛﺮ ﺿﺪ ﺗﻜﺜﻴﺮي اﺳﭙﻮراﻣﻴﻦ اﺳﺘﻔﺎده ﮔﺮدﻳﺪ‪.‬‬

‫ﻳﺎﻓﺘﻪﻫﺎ‪ :‬ﺗﻚ ﺑﺎﻧﺪ ‪ 25‬ﻛﻴﻠﻮداﻟﺘﻮﻧﻲ ﭘﺲ از اﻧﺠﺎم ‪ ،SDS-PAGE‬ﻧﻤﺎﻳﺎﻧﮕﺮ اﺳﭙﻮراﻣﻴﻦ ﺑﻮد‪ .‬ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎري آن ﻧﻴﺰ ‪ 800‬واﺣﺪ ﻣﻬﺎري در ﻣﻴﻠﻲﮔﺮم در دﻗﻴﻘﻪ ﺑﻪ دﺳﺖ آﻣﺪ‪.‬‬
‫ﻧﺘﺎﻳﺞ روش ‪ ،MTT‬ﻣﻴﺰان ‪ (IC50) Inhibitory concentration50‬ﺑﺮاي ‪ 24‬و ‪ 48‬ﺳﺎﻋﺖ داراي ﺗﻔﺎوت ﻣﻌﻨﻲداري ﺑﻮد )‪.(P < 0/010‬‬

‫ﻧﺘﻴﺠﻪﮔﻴﺮي‪ :‬روش ﺗﻚ ﺳﺘﻮﻧﻲ ﺗﻌﻮﻳﺾ ﻳﻮﻧﻲ و ﺷﻴﺐ ﻧﻤﻜﻲ ﭘﻴﻮﺳﺘﻪ ﺑﺮاي ﺧﺎﻟﺺﺳﺎزي اﺳﭙﻮراﻣﻴﻦ ﻣﻮﻓﻘﻴﺖآﻣﻴﺰ ﺑﻮد و ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ اﺛﺮ ﺿﺪ ﺗﻜﺜﻴﺮي آن ﺑﺮ روي ﺳﻠﻮلﻫﺎي‬
‫‪ ،MCF-7‬اﻣﻴﺪ اﺳﺖ ﺑﺘﻮان در آﻳﻨﺪه از اﻳﻦ روش ﺑﺮاي ﺗﺨﻠﻴﺺ و اﺳﺘﻔﺎده در درﻣﺎن ﺑﻬﺮه ﺑﺮد‪.‬‬

‫واژﮔﺎن ﻛﻠﻴﺪي‪ ،Ipomoea batatas :‬ﺧﺎﻟﺺﺳﺎزي‪ ،‬ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ‪ ،‬اﻟﻜﺘﺮوﻓﻮرز ﭘﻠﻲاﻛﺮﻳﻞ آﻣﻴﺪ‪ ،‬ﺳﺮﻃﺎن ﭘﺴﺘﺎن‬

‫ارﺟﺎع‪ :‬ﻗﻴﻮﻣﻴﺎن ﻣﺮﺿﻴﻪ‪ ،‬ﻋﻠﻲ ﻫﺎﺷﻤﻲ ﻋﺎدﻟﻪ‪ ،‬اﻣﻴﺪي اﺳﻜﻮﻳﻲ ﻣﺮﻳﻢ‪ ،‬ﻧﻴﻜﻮﻛﺎر اﻳﺮج‪ ،‬ﻛﺒﻴﺮي آزاده‪ .‬ﺑﻬﻴﻨﻪﺳﺎزي ﺗﺨﻠﻴﺺ اﺳﭙﻮراﻣﻴﻦ ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‬
‫]‪ [Ipomoea Batatas (L.) Lam‬و ﺑﺮرﺳﻲ اﺛﺮ ﺿﺪ ﺗﻜﺜﻴﺮي آن ﺑﺮ ﺳﻠﻮلﻫﺎي ﺳﺮﻃﺎن ﭘﺴﺘﺎن‪ ،‬ردهي ‪ .MCF-7‬ﻣﺠﻠﻪ داﻧﺸﻜﺪه ﭘﺰﺷﻜﻲ‬
‫اﺻﻔﻬﺎن ‪1396‬؛ ‪565-570 :(430) 35‬‬

‫ﺳﺮﻃﺎن ﭘﺴﺘﺎن از ﺟﻤﻠﻪ ﺷﺎﻳﻊﺗﺮﻳﻦ ﺳﺮﻃﺎنﻫﺎ در ﺧﺎﻧﻢﻫﺎ ﻣـﻲﺑﺎﺷـﺪ‬ ‫ﻣﻘﺪﻣﻪ‬

‫ﻛﻪ روشﻫﺎي ﻣﺘﺪاول درﻣﺎن‪ ،‬ﻣﺎﻧﻨﺪ اﺳﺘﻔﺎده از ﺷﻴﻤﻲدرﻣﺎﻧﻲ ﺑﺎ دز ﺑـﺎﻻ‪،‬‬
‫ﭘﺲ از ﺑﺮداﺷﺖ ﺗﻮده ﻋﻼوه ﺑﺮ ﻫﺰﻳﻨﻪﻫﺎي ﻫﻨﮕﻔﺘﻲ ﻛﻪ ﺑﻪ دﻧﺒﺎل دارد‪ ،‬ﺑﻪ‬
‫ﻃﻮر ﻣﻌﻤﻮل داراي ﻋﻮارض ﻣﺨﺘﻠﻒ و ﻧﺎﺧﻮﺷﺎﻳﻨﺪي ﻫﻤﭽﻮن ﻣﻘﺎوﻣـﺖ‬
‫ﺑﻪ دارو اﺳﺖ‪ .‬از اﻳﻦ رو‪ ،‬ﻣﺤﻘﻘﺎن ﺑﻪ دﻧﺒﺎل ﻳﺎﻓﺘﻦ راه ﺣﻞﻫـﺎي ﺳـﺎده و‬
‫ارزانﺗﺮ و اﻏﻠﺐ ﺑﺎ ﻣﻨﺸﺄ ﮔﻴﺎﻫﻲ ﻫﺴﺘﻨﺪ )‪.(3‬‬
‫ﭘﺮوﺗﺌﺎزﻫــﺎ در اﻳﺠــﺎد ﻓﺮاﻳﻨــﺪﻫﺎي ﻣﺘﻔــﺎوﺗﻲ از ﺟﻤﻠــﻪ آﻏــﺎز و ﭘﻴﺸــﺮﻓﺖ‬
‫ﺑﻴﻤﺎريﻫﺎﻳﻲ ﻣﺎﻧﻨﺪ ﺳﺮﻃﺎن دﺧﺎﻟﺖ دارﻧﺪ‪ .‬ﭘﺮوﺗﺌﺎزﻫﺎي ﻣﺘﻔﺎوﺗﻲ ﻧﻈﻴﺮ ﺳﺮﻳﻦ‪،‬‬
‫ﺳﻴﺴﺘﺌﻴﻦ و ﻣﺘﺎﻟﻮﭘﺮوﺗﺌﻴﻨﺎزﻫﺎ‪ ،‬در ﻣﺘﺎﺳﺘﺎز ﺳﺮﻃﺎن ﻧﻘﺶ ﻛﻠﻴﺪي اﻳﻔﺎ ﻣﻲﻛﻨﻨﺪ و‬
‫ﺑﻪ ﻫﻤﻴﻦ دﻟﻴﻞ‪ ،‬ﺑﺎﻳﺪ ﺑﻪ ﺷﺪت ﻛﻨﺘﺮل ﮔﺮدﻧﺪ )‪ .(1‬ﺗﻌﺎدل ﻣﻴﺎن ﻣﻴﺰان ﭘﺮوﺗﺌﺎز و‬
‫ﻣﻬﺎر ﻛﻨﻨﺪهي آن‪ ،‬در ﻫﻤﻮﺳﺘﺎز ﺳﻠﻮل ﻧﻘﺶ ﺣﻴﺎﺗﻲ دارد )‪.(2‬‬

‫‪ -1‬ﻣﺮﻛﺰ ﺗﺤﻘﻴﻘﺎت زﻳﺴﺖ ﻓﻦآوري ﭘﺰﺷﻜﻲ‪ ،‬داﻧﺸﻜﺪهي ﭘﺮﺳﺘﺎري‪ ،‬ﻣﺎﻣﺎﻳﻲ و ﭘﻴﺮاﭘﺰﺷﻜﻲ‪ ،‬داﻧﺸﮕﺎه ﻋﻠﻮم ﭘﺰﺷﻜﻲ ﮔﻴﻼن‪ ،‬رﺷﺖ‪ ،‬اﻳﺮان‬
‫‪ -2‬ﻣﺮﺑﻲ‪ ،‬ﻣﺮﻛﺰ ﺗﺤﻘﻴﻘﺎت زﻳﺴﺖ ﻓﻦآوري ﭘﺰﺷﻜﻲ‪ ،‬داﻧﺸﻜﺪهي ﭘﺮﺳﺘﺎري‪ ،‬ﻣﺎﻣﺎﻳﻲ و ﭘﻴﺮاﭘﺰﺷﻜﻲ‪ ،‬داﻧﺸﮕﺎه ﻋﻠﻮم ﭘﺰﺷﻜﻲ ﮔﻴﻼن‪ ،‬رﺷﺖ‪ ،‬اﻳﺮان‬
‫‪ -3‬داﻧﺸﻴﺎر‪ ،‬ﻣﺮﻛﺰ ﺗﺤﻘﻴﻘﺎت زﻳﺴﺖ ﻓﻦآوري ﭘﺰﺷﻜﻲ‪ ،‬داﻧﺸﻜﺪهي ﭘﺮﺳﺘﺎري‪ ،‬ﻣﺎﻣﺎﻳﻲ و ﭘﻴﺮاﭘﺰﺷﻜﻲ‪ ،‬داﻧﺸﮕﺎه ﻋﻠﻮم ﭘﺰﺷﻜﻲ ﮔﻴﻼن‪ ،‬رﺷﺖ‪ ،‬اﻳﺮان‬
‫‪ -4‬اﺳﺘﺎدﻳﺎر‪ ،‬ﻣﺮﻛﺰ ﺗﺤﻘﻴﻘﺎت زﻳﺴﺖ ﻓﻦآوري ﭘﺰﺷﻜﻲ‪ ،‬داﻧﺸﻜﺪهي ﭘﺮﺳﺘﺎري‪ ،‬ﻣﺎﻣﺎﻳﻲ و ﭘﻴﺮاﭘﺰﺷﻜﻲ‪ ،‬داﻧﺸﮕﺎه ﻋﻠﻮم ﭘﺰﺷﻜﻲ ﮔﻴﻼن‪ ،‬رﺷﺖ‪ ،‬اﻳﺮان‬
‫‪Email: kabiri@gums.ac.ir‬‬ ‫ﻧﻮﻳﺴﻨﺪهي ﻣﺴﺆول‪ :‬آزاده ﻛﺒﻴﺮي‬

‫‪565‬‬ ‫ﻣﺠﻠﻪ داﻧﺸﻜﺪه ﭘﺰﺷﻜﻲ اﺻﻔﻬﺎن – ﺳﺎل ‪ / 35‬ﺷﻤﺎرهي ‪ /430‬ﻫﻔﺘﻪي دوم ﺗﻴﺮ ‪1396‬‬

‫‪www.mui.ac.ir‬‬
‫ﻣﺮﺿﻴﻪ ﻗﻴﻮﻣﻴﺎن و ﻫﻤﻜﺎران‬
‫‪ 3000x g‬ﺑﻪ ﻣﺪت ‪ 10‬دﻗﻴﻘﻪ ﺳـﺎﻧﺘﺮﻳﻔﻴﻮژ ﮔﺮدﻳـﺪ‪ .‬ﻫـﻢ ﺣﺠـﻢ ﻣﺤﻠـﻮل‬
‫روﻳﻲ‪ ،‬ﺳﻮﻟﻔﺎت آﻣﻮﻧﻴﻮم ‪ 60‬درﺻﺪ اﺿـﺎﻓﻪ ﮔﺮدﻳـﺪ‪ .‬ﺳـﭙﺲ‪ ،‬ﺑـﺎ ﺷـﺘﺎب‬
‫‪ 3000 x g‬ﺑﻪ ﻣﺪت ‪ 20‬دﻗﻴﻘﻪ ﺳﺎﻧﺘﺮﻳﻔﻴﻮژ ﺷﺪ‪ .‬رﺳﻮب ﺣﺎﺻﻞ در ﺑﺮاﺑـﺮ‬
‫ﺑﺎﻓﺮ دﻳﺎﻟﻴﺰ ﮔﺮدﻳﺪ‪ .‬ﺳﭙﺲ‪ ،‬ﺑﺎ ﺳـﺮﻋﺖ ‪ 200‬ﻣﻴﻜﺮوﻟﻴﺘـﺮ ﺑـﺮ دﻗﻴﻘـﻪ‪ ،‬وارد‬
‫ﺳﺘﻮن ‪(DEAE-Sepharose) Diethylaminoethanol-Sepharose‬‬
‫)‪ 100 × 15) (GE Healthcare‬ﻣﻴﻠﻲﻣﺘﺮ ﻣﺮﺑﻊ( ﺷﺪ ﻛﻪ از ﻗﺒﻞ ﺑﺎ ﺑـﺎﻓﺮ‬
‫ﻣﺘﻌﺎدل ﺷﺪه ﺑـﻮد‪ .‬آن ﮔـﺎه‪ ،‬ﺳـﺘﻮن ﺑـﺎ ﺷـﻴﺐ ﻧﻤﻜـﻲ ﭘﻴﻮﺳـﺘﻪي ‪NaCl‬‬
‫‪ 0/1-0/5‬ﻣﻮﻻر ﺷﺴﺘﺸﻮ داده ﺷﺪ‪ .‬ﻓﺮاﻛﺸﻦﻫﺎي ﺧـﺎرج ﺷـﺪه از ﺳـﺘﻮن‬
‫ﺟﻤﻊآوري ﮔﺮدﻳﺪ و ﺑﺎ اﺳﺘﻔﺎده از دﺳﺘﮕﺎه اﺳﭙﻜﺘﺮوﻣﺘﺮي در ﻃﻮل ﻣـﻮج‬
‫‪ 280‬ﻧﺎﻧﻮﻣﺘﺮ ﺧﻮاﻧﺪه و ﻛﺮوﻣﺎﺗﻮﮔﺮام رﺳﻢ ﺷﺪ‪.‬‬
‫ﺗﻌﻴﻴﻦ ﺧﻠﻮص ﭘﺮوﺗﺌﻴﻦ اﺳﺘﺨﺮاج ﺷﺪه ﺑﻪ روش ‪:SDS-PAGE‬‬
‫ﺑﺮاي ﺑﺮرﺳﻲ و ﺗﻌﻴﻴﻦ درﺟﻪي ﺧﻠﻮص ﭘﺮوﺗﺌﻴﻦ در ﻧﻤﻮﻧﻪﻫﺎي ﻣﺨﺘﻠـﻒ‬
‫ﺣﺎﺻــﻞ از ﻛﺮوﻣــﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌــﻮﻳﺾ ﻳــﻮن‪ ،‬از روش ‪SDS-PAGE‬‬
‫)‪(Sodium dodecyl sulfate polyacrylamide gel electrophoresis‬‬
‫اﺳﺘﻔﺎده ﮔﺮدﻳﺪ‪ .‬ﺑﻌﺪ از آﻣﺎدهﺳﺎزي و ﻧﺼﺐ ﻗﻄﻌﺎت دﺳﺘﮕﺎه اﻟﻜﺘﺮوﻓﻮرز‬
‫)‪ ،(BioRad‬ﻣﻮاد ژل ﭘﺎﻳﻴﻦ )ﺗﻔﻜﻴﻚ ﻛﻨﻨﺪه( ﻃﺒـﻖ دﺳـﺘﻮراﻟﻌﻤﻞ ﻛﻴـﺖ‬
‫)ﺳــﻴﺘﻮﻣﺘﻴﻦ ژن( ﺑــﺎ ﻏﻠﻈــﺖ ‪ 12/5‬درﺻــﺪ ﺑــﺎ ﻳﻜــﺪﻳﮕﺮ ﻣﺨﻠــﻮط و در‬
‫ﺻﻔﺤﻪي ﻣﺨﺼﻮص اﻟﻜﺘﺮوﻓﻮرز رﻳﺨﺘﻪ ﺷﺪ‪ .‬ﺑﻌﺪ از ﺑﺴـﺘﻪ ﺷـﺪن ﻛﺎﻣـﻞ‬
‫ژل ﭘﺎﻳﻴﻦ‪ ،‬ژل ﺑﺎﻻ )ردﻳـﻒ ﻛﻨﻨـﺪه( ﻧﻴـﺰ ﺑـﺎ ﻏﻠﻈـﺖ ‪ 5‬درﺻـﺪ و ﻃﺒـﻖ‬
‫دﺳﺘﻮراﻟﻌﻤﻞ ﻛﻴﺖ ﺳﺎﺧﺘﻪ و روي ژل ﭘﺎﻳﻴﻦ رﻳﺨﺘﻪ ﺷﺪ‪ .‬ﺑﻌﺪ از رﻳﺨـﺘﻦ‬
‫ژل ﺑﺎﻻ‪ ،‬ﺷﺎﻧﻪي ﻣﺮﺑﻮط ﺑﻪ ﭼﺎﻫﻚ ﻧﻴﺰ در ژل ﻓﺮو ﺑﺮده وزﻣﺎن داده ﺷـﺪ‬
‫ﺗﺎ ژل ﺑﺒﻨﺪد‪ .‬ﻣﻴﺰان ﻻزم از ﻫﺮ ﻧﻤﻮﻧﻪي ﭘﺮوﺗﺌﻴﻨـﻲ و ﻧﺸـﺎﻧﮕﺮ ﭘﺮوﺗﺌﻴﻨـﻲ‪،‬‬
‫درون ﭼﺎﻫﻚﻫﺎ رﻳﺨﺘﻪ ﺷﺪ و ﺳﭙﺲ‪ ،‬اﻟﻜﺘﺮوﻓﻮرز ﺑـﺎ اﺧـﺘﻼف ﭘﺘﺎﻧﺴـﻴﻞ‬
‫‪ 100‬وات اﻧﺠــﺎم ﮔﺮدﻳــﺪ‪ .‬ﺳــﭙﺲ‪ ،‬ژل در ﻣﺤﻠــﻮل رﻧــﮓ ﻛــﻪ ﺣــﺎوي‬
‫‪ Coomassie Blue R-250‬و ﻣﺘﺎﻧﻮل ﺑﻮد‪ ،‬ﻗﺮار داده ﺷﺪ‪.‬‬
‫ژل ﺑﺎ آب ﻣﻘﻄﺮ ﺷﺴﺘﺸﻮ و در ﻣﺤﻠﻮل رﻧﮓﺑﺮ ﻗﺮار داده ﺷﺪ‪ .‬ﺟﻬﺖ‬
‫رﻧﮓزداﻳﻲ ﻛﺎﻣﻞ ژل و ﻫﻮﻳﺪا ﺷﺪن ﺑﺎﻧﺪﻫﺎي ﭘﺮوﺗﺌﻴﻨﻲ ﻣﺠﺰا‪ ،‬در ﻃﻴـﻒ‬
‫زﻣﺎﻧﻲ ‪ 48‬ﺳﺎﻋﺘﻪ‪ ،‬ﭼﻨﺪﻳﻦ ﻣﺮﺗﺒﻪ ﻣﺤﻠﻮل رﻧﮓﺑﺮ ﺗﻌﻮﻳﺾ ﺷﺪ‪.‬‬
‫اﻧﺪازهﮔﻴﺮي ﻣﻴﺰان ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎر ﻛﻨﻨﺪهﮔﻲ ﺗﺮﻳﭙﺴـﻴﻦ اﺳـﭙﻮراﻣﻴﻦ‬
‫ﺧﺎﻟﺺ ﺳﺎزي ﺷﺪه‪ :‬ﻣﻴﺰان ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎري اﺳـﭙﻮراﻣﻴﻦ اﺳـﺘﺨﺮاج ﺷـﺪه‬
‫ﻣﻄﺎﺑﻖ روش ‪ Laskowaski-Takanara‬اﻧﺪازهﮔﻴﺮي ﺷـﺪ‪ .‬اﻳـﻦ روش‬
‫ﻣﺒﺘﻨﻲ ﺑﺮ ﻣﻤﺎﻧﻌﺖ از اﻓﺰاﻳﺶ رﻧﮓ ﺣﺎﺻﻞ از ﻋﻤﻞ ﺗﺮﻳﭙﺴﻴﻦ ﺑﺮ ﻣﺤﻠـﻮل‬
‫‪ (BAEE) Nα-Benzoyl-L-arginine ethyl ester‬اﺳـــﺖ‪ .‬اﺛـــﺮ‬
‫اﺳﺘﺮازي ﺗﺮﻳﭙﺴﻴﻦ ﺑﺮ ﺳﻮﺑﺴـﺘﺮاي ‪ BAEE‬ﺑـﺎ اﻓـﺰاﻳﺶ ﺟـﺬب ﻫﻤـﺮاه‬
‫اﺳﺖ‪ .‬در ﺻﻮرت اﺿـﺎﻓﻪ ﻛـﺮدن ﻣﻬـﺎر ﻛﻨﻨـﺪهي ﺗﺮﻳﭙﺴـﻴﻦ‪ ،‬از اﻓـﺰاﻳﺶ‬
‫ﺟﺬب ﺑﻪ ﻋﻠﺖ ﻣﻬﺎر ﺷﺪن ﻓﻌﺎﻟﻴﺖ اﺳﺘﺮازي ﺗﺮﻳﭙﺴﻴﻦ ﻣﻤﺎﻧﻌﺖ ﺑﻪ ﻋﻤـﻞ‬
‫ﻣﻲآﻳﺪ )‪ .(13‬در اﻳﻦ روش‪ ،‬ﻫﺮ واﺣﺪ ﻣﻬﺎري ﺑﺮاﺑﺮ ﺑﺎ ﻛﺎﻫﺶ ﺟﺬب ﺑـﻪ‬
‫اﻧﺪازهي ‪ 0/001‬در ﻫﺮ دﻗﻴﻘﻪ در ﻃﻮل ﻣﻮج ‪ 253‬ﻧﺎﻧﻮﻣﺘﺮ ﻣﻲﺑﺎﺷﺪ‪.‬‬
‫ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎري آﻧﺰﻳﻢ ﻣﻬﺎر ﻛﻨﻨﺪه در ﻳﻚ ﻣﻴﻠﻲﮔﺮم ﭘﺮوﺗﺌﻴﻦ ﺧﺎﻟﺺ‬
‫ﻣﺠﻠﻪ داﻧﺸﻜﺪه ﭘﺰﺷﻜﻲ اﺻﻔﻬﺎن – ﺳﺎل ‪ / 35‬ﺷﻤﺎرهي ‪ /430‬ﻫﻔﺘﻪي دوم ﺗﻴﺮ ‪  1396‬‬
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‫ﺑﻬﻴﻨﻪﺳﺎزي ﺗﺨﻠﻴﺺ اﺳﭙﻮراﻣﻴﻦ ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‬
‫ﮔﻴﺎﻫﺎن ﺑﺮاي دﻓﺎع از ﺧﻮد در ﺑﺮاﺑﺮ آﻓﺎت )ﺣﺸـﺮات( ﺑـﻪ ﺳـﺮﻋﺖ‬
‫ﺷﺮوع ﺑﻪ ﺳﺎﺧﺖ اﻧﻮاﻋﻲ از ﻣﻬﺎر ﻛﻨﻨﺪﮔﺎن ﭘﺮوﺗﺌﺎزي ﻣﻲﻛﻨﻨـﺪ)‪ .(4‬ﻣﻬـﺎر‬
‫ﻛﻨﻨﺪﮔﺎن ﭘﺮوﺗﺌﺎزي‪ ،‬ﮔﺮوه ﺑﺰرﮔﻲ از ﭘﺮوﺗﺌﻴﻦﻫﺎ ﻫﺴﺘﻨﺪ ﻛﻪ ﻣﻲﺗﻮاﻧﻨـﺪ ﺑـﺎ‬
‫ﺷﻜﺴﺘﻦ رﺷﺘﻪﻫﺎي ﭘﻠﻲﭘﭙﺘﻴﺪي آﻧﺰﻳﻢﻫﺎ‪ ،‬در ﺑﺮاﺑﺮ آنﻫـﺎ ﻣﻘﺎوﻣـﺖ ﻛﻨﻨـﺪ‪.‬‬
‫ﻣﻬﺎر ﻛﻨﻨـﺪهﻫـﺎي ﭘﺮوﺗﺌـﺎزي را ﻣـﻲﺗـﻮان از ﻣﻨـﺎﺑﻊ ﻣﺨﺘﻠﻔـﻲ از ﺟﻤﻠـﻪ‬
‫ﺑﺨﺶﻫﺎي ﻣﺨﺘﻠﻒ ﮔﻴﺎﻫﺎن ﺟﺪاﺳﺎزي ﻛﺮد )‪.(4-5‬‬
‫ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ ﺑﺎ ﻧﺎم ﻋﻠﻤﻲ ])‪[Ipomoea batatas (L.Lam‬‬
‫ﮔﻴﺎﻫﻲ دوﻟﭙﻪاي اﺳﺖ ﻛﻪ ﺑﻪ ﺻﻮرت ﮔﺴﺘﺮده در ﻧﻮاﺣﻲ آﺳﻴﺎي ﺟﻨـﻮب‬
‫ﺷﺮﻗﻲ )ﻣﻨﺎﻃﻖ اﺳﺘﻮاﻳﻲ( ﻛﺸﺖ ﻣﻲﺷﻮد )‪ (6‬و ﺑﻪ ﻋﻨﻮان ﻏـﺬاي اﻧﺴـﺎن‪،‬‬
‫ﺧﻮراك دام و ﻣﺼﺎرف ﺻﻨﻌﺘﻲ ﻛﺎرﺑﺮد دارد )‪ .(7‬ﺗﺤﻘﻴﻘﺎت ﻧﺸﺎن دادهاﻧﺪ‬
‫ﻛﻪ ‪ 60-80‬درﺻﺪ ﭘـﺮوﺗﺌﻴﻦ ﻣﺤﻠـﻮل در ﺑﺨـﺶ رﻳﺸـﻪي ﺳـﻴﺐزﻣﻴﻨـﻲ‬
‫ﺷﻴﺮﻳﻦ را ﻣﻬﺎر ﻛﻨﻨﺪهي ﺗﺮﻳﭙﺴﻴﻦ )اﺳـﭙﻮراﻣﻴﻦ( ﺗﺸـﻜﻴﻞ ﻣـﻲدﻫـﺪ )‪.(8‬‬
‫اﺳﭙﻮراﻣﻴﻦ‪ ،‬ﺧﺎﺻﻴﺖ ﺿﺪ ﺗﻜﺜﻴﺮي و اﻟﻘﺎ ﻛﻨﻨﺪﮔﻲ آﭘﻮﭘﺘﻮز را در در ﭼﻨﺪ‬
‫ﻧﻮع ردهي ﺳﻠﻮﻟﻲ ﺳﺮﻃﺎﻧﻲ )زﺑﺎن‪ ،‬ﻛﻮﻟﻮن و ‪ (...‬از ﺧﻮد ﻧﺸﺎن داده اﺳﺖ‬
‫)‪.(9-10‬‬
‫روشﻫﺎي ﻣﺨﺘﻠﻔﻲ ﺑﺮاي ﺟﺪاﺳﺎزي اﺳﭙﻮراﻣﻴﻦ ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‬
‫و ﺗﻌﻴﻴﻦ ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎر ﻛﻨﻨﺪﮔﻲ ﺗﺮﻳﭙﺴﻴﻦ ﻣﻮرد اﺳﺘﻔﺎده ﻗﺮار ﮔﺮﻓﺘﻪ اﺳﺖ‪.‬‬
‫ﺑﺮاي ﻣﺜﺎل‪ ،‬روشﻫﺎي دو ﺳﺘﻮﻧﻲ ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌﻮﻳﺾ ﻳﻮﻧﻲ و ﺳﭙﺲ‬
‫ژل ﻛﺮوﻣــﺎﺗﻮﮔﺮاﻓﻲ )‪ (10-11‬و ﻳــﺎ ﻛﺮوﻣــﺎﺗﻮﮔﺮاﻓﻲ ﺗﻤــﺎﻳﻠﻲ )‪ (12‬از‬
‫ﺟﻤﻠﻪي روشﻫﺎي ﭼﻨﺪ ﻣﺮﺣﻠﻪاي ﻫﺴـﺘﻨﺪ و ﺳـﺒﺐ اﻓـﺰاﻳﺶ ﻫﺰﻳﻨـﻪي‬
‫اﺳــﺘﺨﺮاج ﻣــﻲﮔﺮدﻧــﺪ‪ .‬در ﻣﻄﺎﻟﻌــﻪي ﺣﺎﺿــﺮ‪ ،‬از روش ﺗــﻚ ﺳــﺘﻮﻧﻲ‬
‫ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌﻮﻳﺾ ﻳﻮﻧﻲ ﺑﺮاي ﺧﺎﻟﺺﺳﺎزي اﺳﭙﻮراﻣﻴﻦ از رﻳﺸـﻪي‬
‫ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ اﺳـﺘﻔﺎده ﺷـﺪ و ﻓﻌﺎﻟﻴـﺖ ﻣﻬـﺎر ﻛﻨﻨـﺪﮔﻲ آن ﺑـﻪ روش‬
‫‪ Laskowaski-Takanara‬اﻧﺪازهﮔﻴﺮي ﮔﺮدﻳﺪ‪ .‬در ﭘﺎﻳﺎن‪ ،‬اﺛﺮ ﺿﺪ ﺗﻜﺜﻴـﺮي‬
‫اﺳﭙﻮراﻣﻴﻦ ﺑﺮاي اوﻟﻴﻦ ﺑﺎر ﺑﺮ ﺳﻠﻮلﻫﺎي ﺳﺮﻃﺎﻧﻲ ﭘﺴﺘﺎن ﺑﺮرﺳﻲ ﺷﺪ‪.‬‬
‫روشﻫﺎ‬
‫ﺧﺎﻟﺺﺳﺎزي اﺳﭙﻮراﻣﻴﻦ از ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‪ :‬ﺗﻌﺪادي ﺳﻴ ﺐ زﻣﻴﻨ ﻲ‬
‫ﺷﻴ ﺮﻳ ﻦ از ﻓﺮوﺷﮕﺎه ﻣﺤﻠ ﻲ در ﺗﻬﺮان ﺧﺮﻳـ ﺪاري ﮔﺮدﻳـﺪ ‪ .‬از رﻳﺸـﻪ‬
‫ﻫﺎي ﺳﻴ ﺐ زﻣﻴﻨ ﻲ ﺷﻴ ﺮﻳ ﻦ ﭘﺲ از ﺷﺴﺘﺸﻮ‪ ،‬ﮔـﺮﻓﺘﻦ ﭘﻮﺳـﺖ و ﺧـﺮد‬
‫ﻛﺮدن ﺑﺎ دﺳﺘﮕﺎه آب ﻣﻴﻮهﮔﻴ ﺮي ‪ ،‬ﻋﺼﺎره ي آﺑ ﻲ ﺗﻬﻴﻪ ﮔﺮدﻳ ﺪ ‪ .‬ﻋﺼﺎره‬
‫ي آﺑــ ﻲ ﺧــﺎم ‪ ،‬ﺑﻼﻓﺎﺻــﻠﻪ ﺑــﺎ ‪ 4‬ﺣﺠــﻢ ﺑــﺎﻓﺮ ‪ 50‬ﻣﻴﻠــ ﻲ ﻣــﻮﻻر‬
‫‪(Tris-HCL) Trisaminomethane-‬‬ ‫‪Hydrogen‬‬ ‫‪chloride‬‬
‫)‪ (pH = 7/6‬ﻛﻪ ﺣﺎوي ‪ 1‬ﻣﻴﻠﻲﻣﻮﻻر ‪Ethylenediaminetetraacetic acid‬‬
‫)‪ 0/1 ،(EDTA‬ﻣــﻮﻻر ‪ (NaCl) Sodium chloride‬و ‪0/1‬درﺻــﺪ‬
‫)‪ W/V‬ﻳﺎ ‪ (Weight/Volume‬آﺳﻜﻮرﺑﻴﻚ اﺳـﻴﺪ ﻣﺨﻠـﻮط ﮔﺮدﻳـﺪ )از‬
‫اﻳﻦ ﺟﺎ ﺑﻪ ﺑﻌﺪ در ﺑﺨﺶ ﺧﺎﻟﺺﺳﺎزي ﺗﺮﻛﻴﺐ ﺑﺎﻓﺮ ﻫﻤﻴﻦ ﻣﻮارد ﺑﻪ ﺟـﺰ‬
‫آﺳﻜﻮرﺑﻴﻚ اﺳﻴﺪ ذﻛﺮ ﺷﺪه اﺳﺖ(‪ .‬ﻣﺨﻠـﻮط ﺣﺎﺻـﻞ‪ ،‬از ﻛﺎﻏـﺬ ﺻـﺎﻓﻲ‬
‫واﺗﻤﻦ ﻋﺒﻮر داده ﺷﺪ‪ .‬ﺳﭙﺲ‪ ،‬در دﻣﺎي ‪ 40‬درﺟﻪي ﺳﺎﻧﺘﻲﮔﺮاد ﺑﺎ ﺷﺘﺎب‬
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 ‫ﻣﺮﺿﻴﻪ ﻗﻴﻮﻣﻴﺎن و ﻫﻤﻜﺎران‬
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‫آﻧﺎﻟﻴﺰ آﻣﺎري‪ :‬ﺑﺮاي آﻧﺎﻟﻴﺰ و ﻣﻘﺎﻳﺴﻪي دادهﻫﺎ از آزﻣـﻮن ‪ t‬اﺳـﺘﻔﺎده‬
‫ﮔﺮدﻳــﺪ‪ .‬آﻧــﺎﻟﻴﺰ دادهﻫــﺎ ﺑــﺎ اﺳــﺘﻔﺎده از ﻧــﺮماﻓــﺰار ‪ SPSS‬ﻧﺴــﺨﻪي ‪19‬‬
‫)‪ (version 19, SPSS Inc., Chicago, IL‬ﺻــﻮرت ﮔﺮﻓــﺖ و‬
‫‪ P < 0/050‬ﺑﻪ ﻋﻨﻮان ﺳﻄﺢ ﻣﻌﻨﻲداري در ﻧﻈﺮ ﮔﺮﻓﺘﻪ ﺷﺪ‪ .‬اﻃﻼﻋﺎت ﺑﻪ‬
‫ﺻﻮرت ﻣﻴﺎﻧﮕﻴﻦ ‪ ±‬اﻧﺤﺮاف ﻣﻌﻴﺎر اراﻳﻪ ﺷﺪه اﺳﺖ‪.‬‬
‫ﻳﺎﻓﺘﻪﻫﺎ‬
‫اﺳﺘﺨﺮاج و ﺧﺎﻟﺺﺳﺎزي اﺳﭙﻮراﻣﻴﻦ از رﻳﺸﻪ ي ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‪:‬‬
‫ﻣﺤﻠﻮلﻫـﺎي ﺑـﻪ دﺳـﺖ آﻣـﺪه از ﺳـﺘﻮن ﻛﺮوﻣـﺎﺗﻮﮔﺮاﻓﻲ در ﻟﻮﻟـﻪﻫـﺎي‬
‫‪ 3‬ﻣﻴﻠﻲﻟﻴﺘﺮي روي ﻳﺦ ﺟﻤﻊآوري ﮔﺮدﻳﺪ و ﺑﺎ اﺳـﭙﻜﺘﺮوﻓﺘﻮﻣﺘﺮ ﺧﻮاﻧـﺪه‬
‫ﺷﺪﻧﺪ‪ .‬ﻛﺮوﻣﺎﺗﻮﮔﺮام رﺳﻢ ﮔﺮدﻳﺪ )ﺷﻜﻞ ‪ .(1‬در ﻛﺮوﻣﺎﺗﻮﮔﺮام‪ ،‬اﺑﺘﺪا ﻳﻚ‬
‫ﻗﻠﻪي ﺑﺰرگ ﻗﺮار دارد ﻛﻪ ﻧﺎﺧﺎﻟﺼﻲﻫﺎ ﻫﺴﺘﻨﺪ و ﺑـﻪ ﺗـﺪرﻳﺞ‪ ،‬ﺑـﺎ اﺿـﺎﻓﻪ‬
‫ﻛﺮدن ﺑﺎﻓﺮ ﺷﺴﺘﺸﻮ و ﺟﺪا ﻣﻲﺷﻮﻧﺪ‪ .‬ﭘﺲ از آن‪ ،‬ﺑﺎ اﺳـﺘﻔﺎده از ﮔﺮادﻳـﺎن‬
‫ﻧﻤﻜﻲ ﺧﻄﻲ‪ ،‬ﻗﻠﻪي دوم ﻣﺮﺑﻮط ﺑﻪ اﺳﭙﻮراﻣﻴﻦ ﻧﻤﺎﻳﺶ داده ﻣﻲﺷﻮد‪.‬‬
‫)‪ABS(280nm‬‬
‫‪7‬‬ ‫‪06‬‬
‫‪NaCl‬‬ ‫)‪concentration(M‬‬
‫‪(M) NaCl‬‬ ‫ﻏﻠﻈﺖ‬
‫‪6‬‬
‫‪05‬‬
‫ﻏﻠﻈﺖ ‪(M)NaCl‬‬
‫‪5‬‬
‫)‪ABS (280nm‬‬
‫‪04‬‬
‫‪4‬‬
‫‪03‬‬
‫‪3‬‬
‫‪02‬‬
‫‪2‬‬
‫‪1‬‬ ‫‪01‬‬
‫‪0‬‬ ‫‪0‬‬
‫‪0‬‬ ‫‪20‬‬ ‫‪40‬‬ ‫‪60‬‬
‫)‪Fraction (3ml‬‬
‫ﺷﻜﻞ ‪ .1‬ﻛﺮوﻣﺎﺗﻮﮔﺮام ﺣﺎﺻﻞ از ﻋﺒﻮر ﻋﺼﺎرهي ﺧﺎم ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‪ ،‬از‬
‫ﺳﺘﻮن ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ‪Diethylaminoethanol-Sepharose‬‬
‫)‪ ،(DEAE-Sepharose‬دو ﻗﻠﻪ ﻣﺸﺎﻫﺪه ﻣﻲﮔﺮدد‪ .‬ﻗﻠﻪي اول ﻣﺮﺑﻮط ﺑﻪ‬
‫ﻧﺎﺧﺎﻟﺼﻲﻫﺎ ﻣﻲﺑﺎﺷﺪ‪ .‬ﻗﻠﻪي دوم ﻛﻪ ﭘﺲ از ﺷﺴﺘﺸﻮي ﺳﺘﻮن ﺑﺎ ﺷﻴﺐ ﻧﻤﻜﻲ‬
‫ﺧﻄﻲ ﻣﺸﺎﻫﺪه ﻣﻲﮔﺮدد‪ ،‬ﻣﺮﺑﻮط ﺑﻪ اﺳﭙﻮراﻣﻴﻦ اﺳﺖ‪.‬‬
‫ﺗﺄﻳﻴﺪ ﺧﻠﻮص اﺳﭙﻮراﻣﻴﻦ اﺳﺘﺨﺮاج ﺷﺪه ﺑﺎ روش ‪:SDS-PAGE‬‬
‫ﺑﺮاي ﺑﺮرﺳﻲ و ﻣﻘﺎﻳﺴﻪي ﻣﻴـﺰان ﺧﻠـﻮص اﺳـﭙﻮراﻣﻴﻦ ﺗﺨﻠـﻴﺺ ﺷـﺪه‪،‬‬
‫اﺳﭙﻮراﻣﻴﻦ ﺑﺎ ﺣﺠﻢ ﺑﺮاﺑﺮ از ﺑﺎﻓﺮ‪ ،‬ﻣﺨﻠﻮط و در ﺣﻀﻮر ﺳﺪﻳﻢ دودﺳـﻴﻞ‬
‫ﺳﻮﻟﻔﺎت در ﺷﺮاﻳﻂ اﺣﻴﺎﻳﻲ اﻟﻜﺘﺮوﻓﻮرز ﮔﺮدﻳﺪ‪ .‬ﻃﺒـﻖ ﺷـﻜﻞ ‪ ،2‬ﺳـﺘﻮن‬
‫‪ ،M‬ﻣﻌﺮف ﻧﺸﺎﻧﮕﺮ ﭘﺮوﺗﺌﻴﻨﻲ و ﺳﺘﻮن ‪ ،1‬ﻣﻌﺮف ﻧﻤﻮﻧـﻪاي از ﻋﺼـﺎرهي‬
‫ﺧﺎم اوﻟﻴﻪي اﺳﺘﺨﺮاج ﺷﺪه از ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ اﺳﺖ‪ .‬وﺟﻮد ﺑﺎﻧﺪﻫﺎي‬
‫ﻣﺨﺘﻠﻒ ﺑﺮ روي ژل‪ ،‬ﻧﺸﺎن از وﺟـﻮد ﻧﺎﺧﺎﻟﺼـﻲ در ﻧﻤﻮﻧـﻪي ﺳـﺘﻮن ‪1‬‬
‫اﺳﺖ و ﺳﺘﻮن ‪ ،2‬ﺑﻴﺎﻧﮕﺮ اﺳﭙﻮراﻣﻴﻦ ﺧﺮوﺟﻲ از ﺳـﺘﻮن ﻛﺮوﻣـﺎﺗﻮﮔﺮاﻓﻲ‬
‫ﻣﻲﺑﺎﺷﺪ‪ .‬ﺗﻚ ﺑﺎﻧـﺪ ‪ 25‬ﻛﻴﻠﻮداﻟﺘـﻮﻧﻲ در ﺳـﺘﻮن ‪ ،2‬ﺑﻴـﺎﻧﮕﺮ دﺳـﺘﻴﺎﺑﻲ ﺑـﻪ‬
‫ﭘﺮوﺗﺌﻴﻦ ﺧﺎﻟﺺ اﺳﭙﻮراﻣﻴﻦ اﺳﺖ )‪.(15‬‬
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‫‪www.mui.ac.ir‬‬
‫ﺑﻬﻴﻨﻪﺳﺎزي ﺗﺨﻠﻴﺺ اﺳﭙﻮراﻣﻴﻦ ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‬
‫ﺷﺪه ﺑﺎ اﺳﺘﻔﺎده از ﻓﺮﻣﻮل زﻳﺮ ﻣﺤﺎﺳﺒﻪ ﺷﺪ‪.‬‬
‫‪∆OD253 nm / min × d.f‬‬
‫=‪TIU‬‬
‫‪0.001 × total protein‬‬
‫]‪[UI/mg/min‬‬
‫‪d.f= dilution factor‬‬
‫ﻛﺸـــــﺖ ﺳـــــﻠﻮل‪ :‬ﺳـــــﻠﻮلﻫـــــﺎي ﺳـــــﺮﻃﺎﻧﻲ ردهي‬
‫‪) (MCF-7) Michigan‬اﻫـــــﺪاﻳﻲ از‬ ‫‪cancer‬‬ ‫‪foundation-7‬‬
‫ﭘﮋوﻫﺸﻜﺪهي اﺑﻦﺳﻴﻨﺎ‪ ،‬ﺗﻬﺮان( ﺟﺰء ردهي ﺳﻠﻮﻟﻲ ﺳﺮﻃﺎن ﭘﺴﺘﺎن از ﻧﻮع‬
‫‪ (ER+) Estrogen receptor+‬و ﻣﺘﺎﺳﺘﺎز دﻫﻨﺪه ﻣﻲﺑﺎﺷﻨﺪ‪ .‬ﺳﻠﻮلﻫﺎ در‬
‫ﻣﺤـــﻴﻂ ﻛﺸـــﺖ ‪Roswell Park Memorial Institute-1640‬‬
‫)‪) (RPMI-1640‬اﻳـــﺪهزﻳﺴـــﺖ ﻧﻮﺗﺮﻛﻴـــﺐ( ﺣـــﺎوي ‪ 10‬درﺻـــﺪ‬
‫‪ 1 ،(Gibco) (FBS) Fetal bovine serum‬درﺻﺪ آﻧﺘﻲﺑﻴﻮﺗﻴﻚ ‪Pen-‬‬
‫‪ ،(Gibco) Strep‬در ﺷـــــﺮاﻳﻂ رﻃﻮﺑـــــﺖ ‪ 95‬درﺻـــــﺪ‪ ،‬ﻓﺸـــــﺎر‬
‫‪ (2CO) Carbon dioxide‬ﺑﺮاﺑﺮ ‪ 5‬و دﻣﺎي ‪ 37‬درﺟﻪي ﺳﺎﻧﺘﻲﮔﺮاد ﻛﺸـﺖ‬
‫داده و ﭘﺲ از رﺳﻴﺪن ﺑﻪ ﺗﺮاﻛﻢ ‪ 80‬درﺻﺪ‪ ،‬ﺳﻠﻮلﻫﺎ ﺑﺮاي ﭘﺎﺳﺎژ آﻣﺎده ﺷﺪﻧﺪ‪.‬‬
‫ﺑﺮرﺳﻲ وﺿﻌﻴﺖ رﺷﺪ ﺳﻠﻮلﻫﺎ در ﺣﻀﻮر اﺳﭙﻮراﻣﻴﻦ ﺑـﻪ روش‬
‫‪ :MTT‬ﺑﺮاي ﺑﺮرﺳﻲ اﺛﺮ ﺿﺪ ﺗﻜﺜﻴﺮي اﺳﭙﻮراﻣﻴﻦ و ﺗﻌﻴـﻴﻦ ﻏﻠﻈـﺖ‬
‫ﻣﻬﺎر ﻛﻨﻨـﺪﮔﻲ )‪ Inhibitory concentration50‬ﻳـﺎ ‪ (IC50‬از روش‬
‫‪3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium‬‬
‫‪ (MTT) bromide‬اﺳﺘﻔﺎده ﮔﺮدﻳﺪ )‪.(12 ،14‬‬
‫ﻣﺮاﺣﻞ اﻧﺠﺎم ﺑﻪ ﻧﺤﻮي ﺑﻮد ﻛﻪ ﻣﻄﺎﺑﻖ دﺳﺘﻮراﻟﻌﻤﻞ ﻛﻴﺖ )اﻳﺪهزﻳﺴﺖ‬
‫ﻧﻮﺗﺮﻛﻴﺐ(‪ ،‬ﭘﻮدر ‪ MTT‬در ﻣﺤﻴﻂ ‪ RPMI-1640‬ﺣﻞ ﮔﺮدﻳﺪ‪ .‬ﺑﻪ ﻣﻨﻈـﻮر‬
‫‪80‬‬
‫اﻧﺠﺎم روش ‪ ،MTT‬ﺳﻮﺳﭙﺎﻧﺴﻴﻮﻧﻲ ﺣﺎوي ‪ 7000‬ﺳﻠﻮل در ﻣﺤـﻴﻂ ﻛﺎﻣـﻞ‬
‫ﺑﻪ ﻫﺮ ﭼﺎﻫﻚ ﭘﻠﻴﺖ ‪ 96‬ﺧﺎﻧﻪ اﻓﺰوده ﺷـﺪ‪ .‬ﭘـﺲ از ﮔﺬﺷـﺖ ﻳـﻚ ﺷـﺐ‪،‬‬
‫ﺳﻠﻮلﻫﺎ ﮔﺮوهﺑﻨﺪي ﺷﺪﻧﺪ‪.‬‬
‫ﺑﺮاي ﮔﺮوه ﺷﺎﻫﺪ‪ ،‬اﻧﻜﻮﺑﺎﺳﻴﻮن ﺗﻨﻬﺎ ﺑﺎ ﻣﺤـﻴﻂ ﻛﺸـﺖ ﺑـﺪون ﺳـﺮم‬
‫‪ RPMI-1640‬در ﺷﺮاﻳﻂ زﻣﺎﻧﻲ وﻣﻜﺎﻧﻲ ﻳﻜﺴﺎن ﺑﺎ ﮔﺮوه ﻣـﻮرد اﻧﺠـﺎم‬
‫ﺷــﺪ و در ﮔــﺮوه ﻣــﻮرد‪ ،‬اﻧﻜﻮﺑﺎﺳــﻴﻮن ﺑــﺎ اﺳــﭙﻮراﻣﻴﻦ ﺑــﺎ ﻏﻠﻈــﺖﻫــﺎي‬
‫‪ 20-2000‬ﻣﻴﻜﺮوﮔﺮم‪/‬ﻣﻴﻠﻲﻟﻴﺘﺮ ﻣﺤﻠﻮل در ﻣﺤـﻴﻂ ﻛﺸـﺖ ﺑـﺪون ﺳـﺮم‬
‫‪ RPMI-1640‬اﻧﺠﺎم ﺷـﺪ‪ .‬ﺑﻌـﺪ از اﺗﻤـﺎم زﻣـﺎن اﻧﻜﻮﺑﺎﺳـﻴﻮن )‪ 24‬ﻳـﺎ‬
‫‪ 48‬ﺳﺎﻋﺖ( ﭼﺎﻫﻚﻫﺎ ﺑﺮاي اﻧﺠﺎم روش ‪ MTT‬آﻣﺎده ﺷﺪﻧﺪ‪.‬‬
‫در ﻧﻬﺎﻳﺖ‪ ،‬ﭘﻠﻴﺖ ‪ 96‬ﺧﺎﻧﻪ ﺣـﺎوي ﺳـﻠﻮل ﺑـﺎ اﺳـﺘﻔﺎده از دﺳـﺘﮕﺎه‬
‫‪Enzyme-linked‬‬ ‫‪immunosorbent‬‬ ‫‪assay‬‬ ‫‪reader‬‬
‫)‪ (ELISA reader‬در ﻃﻮل ﻣﻮج ‪ 490‬ﻧﺎﻧﻮﻣﺘﺮ و ﻃـﻮل ﻣـﻮج رﻓـﺮﻧﺲ‬
‫‪ 630‬ﻧــﺎﻧﻮﻣﺘﺮ در ﻣﻘﺎﺑــﻞ ﺑﻼﻧــﻚ ﺧــﻮاﻧﺶ ﺷــﺪ‪ .‬ﭘــﺲ از ﺟﻤــﻊآوري‬
‫اﻃﻼﻋﺎت‪ IC50 ،‬ﻣﺮﺑﻮط ﺑﻪ ﮔﺮوهﻫﺎي ‪ 24‬و ‪ 48‬ﺳﺎﻋﺖ ﻃﺒﻖ ﻓﺮﻣﻮل زﻳـﺮ‬
‫ﺑﻪ دﺳﺖ آﻣﺪ‪.‬‬
‫‪OD of treatment‬‬
‫× ‪(%) cell viability = 100‬‬
‫‪OD of control‬‬
‫ﻣﺠﻠﻪ داﻧﺸﻜﺪه ﭘﺰﺷﻜﻲ اﺻﻔﻬﺎن – ﺳﺎل ‪ / 35‬ﺷﻤﺎرهي ‪ /430‬ﻫﻔﺘﻪي دوم ﺗﻴﺮ ‪1396‬‬
‫ﻣﺮﺿﻴﻪ ﻗﻴﻮﻣﻴﺎن و ﻫﻤﻜﺎران‬ ‫ﺑﻬﻴﻨﻪﺳﺎزي ﺗﺨﻠﻴﺺ اﺳﭙﻮراﻣﻴﻦ ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‬

‫ﺑﺤﺚ‬
‫اﻣﺮوزه‪ ،‬ﻳﺎﻓﺘﻦ روشﻫﺎي ﺑﻬﻴﻨﻪ و ﻛﺎرآﻣـﺪ در ﺧـﺎﻟﺺﺳـﺎزي ﺗﺮﻛﻴﺒـﺎت‬
‫ﮔﻴﺎﻫﻲ داراي ﺧﻮاص درﻣﺎﻧﻲ‪ ،‬اﻫﻤﻴﺖ زﻳﺎدي ﭘﻴﺪا ﻛﺮده اﺳﺖ‪ .‬در ﺳـﺎل‬
‫‪ ،1985‬اﺳــﭙﻮراﻣﻴﻦ ﺑــﺮاي اوﻟــﻴﻦ ﺑــﺎر از ﻋﺼــﺎرهي ﻣﺤﻠــﻮل رﻳﺸــﻪي‬
‫ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ ﺗﺨﻠﻴﺺ ﮔﺮدﻳﺪ و روي ‪ ،SDS-PAGE‬ﺑﻪ ﺻـﻮرت‬
‫ﺗﻚ ﺑﺎﻧﺪ ‪ 25‬ﻛﻴﻠﻮداﻟﺘﻮﻧﻲ ﻣﺸﺎﻫﺪه ﺷﺪ )‪ (15‬ﻛﻪ ﺑﺎ ﻧﺘﺎﻳﺞ ﻣﻄﺎﻟﻌﻪي ﺣﺎﺿﺮ‬
‫ﻫﻢﺧﻮاﻧﻲ دارد‪.‬‬
‫در ﻣﻄﺎﻟﻌﺎت ﻗﺒﻠـﻲ‪ ،‬از روشﻫـﺎي دو ﺳـﺘﻮن ﭘـﻲ در ﭘـﻲ ﻫﻤﺎﻧﻨـﺪ‬
‫اﺳﺘﻔﺎده از ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌﻮﻳﺾ ﻳﻮﻧﻲ ‪ DEAE-Cellulose‬و ﺳﭙﺲ‪،‬‬
‫ﻓﻴﻠﺘﺮاﺳــﻴﻮن ژﻟــﻲ ﺑــﺎ ‪ (10-11 ،15) Sephadex-G75‬و ﻳــﺎ روش‬ ‫ﺷﻜﻞ ‪ .2‬ﺗﺄﻳﻴﺪ ﺧﻠﻮص اﺳﭙﻮراﻣﻴﻦ اﺳﺘﺨﺮاج ﺷﺪه از ﻃﺮﻳﻖ اﻟﻜﺘﺮوﻓﻮرز ﺑﺮ روي ژل‬
‫ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻤﺎﻳﻠﻲ )‪ (12‬ﺑﺮاي اﺳﺘﺨﺮاج اﺳـﭙﻮراﻣﻴﻦ اﺳـﺘﻔﺎده ﺷـﺪه‬ ‫‪Sodium dodecyl sulfate polyacrylamide gel electrophoresis‬‬
‫اﺳﺖ‪ .‬روش ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻤﺎﻳﻠﻲ ﻧﺴﺒﺖ ﺑﻪ ﻛﺮوﻣـﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌـﻮﻳﺾ‬ ‫)‪ .(SDS-PAGE‬ﺳﺘﻮن ‪ ،M‬ﻣﺮﺑﻮط ﺑﻪ ﻧﺸﺎﻧﮕﺮ ﭘﺮوﺗﺌﻴﻨﻲ‪ ،‬ﺳﺘﻮن ‪ 1‬ﻣﺮﺑﻮط‬

‫ﻳﻮﻧﻲ‪ ،‬ﮔﺮان ﺗﺮ و ﻧﻴﺎزﻣﻨﺪ ﻃﺮاﺣﻲ ﻧﺸﺎﻧﮕﺮ ﺧـﺎص ﺑـﺮاي ﺧـﺎﻟﺺﺳـﺎزي‬ ‫ﺑﻪ ﻋﺼﺎرهي ﺧﺎم )ﭘﻴﺶ از ﺑﺮدن روي ﺳﺘﻮن ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ( اﺳﺖ‪ .‬وﺟﻮد‬
‫ﺑﺎﻧﺪﻫﺎي ﻣﺨﺘﻠﻒ‪ ،‬ﻧﻤﺎﻳﺎﻧﮕﺮ ﻧﺎﺧﺎﻟﺼﻲ اﺳﺖ‪ .‬ﺳﺘﻮن ‪ ،2‬ﺧﺮوﺟﻲ ﺳﺘﻮن‬
‫اﺳﺖ )‪ .(16‬از ﻣﺰاﻳﺎي روش ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌﻮﻳﺾ ﻳﻮﻧﻲ‪ ،‬ﻣﻲﺗﻮان ﺑـﻪ‬
‫ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﭘﺲ از اﻓﺰودن ﺷﻴﺐ ﻧﻤﻜﻲ ﺧﻄﻲ ﺑﻪ ﺳﺘﻮن اﺳﺖ ﻛﻪ ﺣﺎﺻﻞ‬
‫ﻣﺪت زﻣـﺎن ﻛﻮﺗـﺎه آﻧـﺎﻟﻴﺰ‪ ،‬ﺣﺴﺎﺳـﻴﺖ زﻳـﺎد ﻧﺴـﺒﺖ ﺑـﻪ ﻣـﺎده در ﺣـﺪ‬
‫ﺗﻚ ﺑﺎﻧﺪ ‪ 25‬ﻛﻴﻠﻮداﻟﺘﻮﻧﻲ اﺳﺖ‪.‬‬
‫ﻣﻴﻜﺮوﮔﺮم در ﻟﻴﺘﺮ‪ ،‬اﺳﺘﻔﺎده از ﻣﻮاد ﺷﻴﻤﻴﺎﻳﻲ ارزان و ﺳﺎﻟﻢ ﺑﺮاي ﻣﺤـﻴﻂ‬
‫زﻳﺴــﺖ و ‪ ...‬اﺷــﺎره ﻛــﺮد )‪ .(17‬اﺳــﺘﺨﺮاج اﺳــﭙﻮراﻣﻴﻦ از رﻳﺸــﻪي‬
‫ﺗﻌﻴﻴﻦ ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎر ﻛﻨﻨﺪﮔﻲ ﺗﺮﻳﭙﺴﻴﻦ و اﺳﭙﻮراﻣﻴﻦ ﺧﺎﻟﺺ ﺷﺪه‪:‬‬
‫ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‪ ،‬ﺑﺎ ﺗﻚ ﺳﺘﻮن ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌﻮﻳﺾ ﻳﻮﻧﻲ ﺑﺎ ﻟﻴﮕﺎﻧﺪ‬
‫ﺳﻨﺠﺶ ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎري اﺳﭙﻮراﻣﻴﻦ ﺧﺎﻟﺺ ﺷﺪه ﺑﻪ روش ﻛﻴﻨﺘﻴﻚ اﻧﺠﺎم‬
‫‪ ،DEAE-Sepharose‬ﺑﺮاي اوﻟﻴﻦ ﺑـﺎر در ﻣﻄﺎﻟﻌـﻪي ﺣﺎﺿـﺮ ﺻـﻮرت‬
‫ﺷﺪ‪ .‬اﺳﭙﻮراﻣﻴﻦ‪ ،‬داراي ‪ 800‬واﺣﺪ ﻣﻬـﺎري ﺗﺮﻳﭙﺴـﻴﻦ در ﻣﻴﻠـﻲﮔـﺮم در‬
‫ﮔﺮﻓﺖ‪ .‬دﻟﻴﻞ ﻣﻮﻓﻘﻴﺖ روش ﺗﻚ ﺳﺘﻮﻧﻲ در ﻣﻄﺎﻟﻌﻪي ﺣﺎﺿﺮ‪ ،‬اﺳﺘﻔﺎده از‬
‫دﻗﻴﻘﻪ ﺑﻮد‪.‬‬
‫ﺷﻴﺐ ﻧﻤﻜﻲ ﭘﻴﻮﺳﺘﻪ )‪ 0/1-0/5‬ﻣـﻮﻻر ‪ (NaCl‬ﺑـﻮد؛ در ﺣـﺎﻟﻲ ﻛـﻪ در‬
‫ﻣﻄﺎﻟﻌﻪي دﻳﮕﺮي‪ ،‬از ﻳﻚ ﻏﻠﻈـﺖ ﻧﻤﻜـﻲ ﻣﺸـﺨﺺ ﻣﺎﻧﻨـﺪ ‪ 0/2‬ﻣـﻮﻻر‬ ‫ﺗﻌﻴـﻴﻦ ‪ IC50‬اﺳـﭙﻮراﻣﻴﻦ ‪ 24‬و ‪ 48‬ﺳـﺎﻋﺖ ﭘـﺲ از ﺗﻴﻤـﺎر ﺑـﺎ‬

‫‪ (10) NaCl‬و ﻳـــﺎ در ﻣﻄﺎﻟﻌـــﻪي دﻳﮕـــﺮي‪ ،‬از ﻏﻠﻈـــﺖ ‪ 2/0‬ﻣـــﻮﻻر‬ ‫ﺳـﻠﻮلﻫـﺎي ‪ :MCF-77‬ﭘـﺲ از ﺧﻮاﻧـﺪن ‪(OD) Optical density‬‬

‫‪ (KCL) Potassium chloride‬اﺳــﺘﻔﺎده )‪ (15‬و ﺳــﭙﺲ‪ ،‬روش ژل‬ ‫ﺳﻠﻮلﻫﺎي ﮔﺮوهﻫﺎي ﺷﺎﻫﺪ و ﻣﻮرد در ‪ 24‬و ‪ 48‬ﺳﺎﻋﺖ و ﻗﺮار دادن آن‬
‫ﻓﻴﻠﺘﺮاﺳﻴﻮن ﺑﻪ ﻛﺎر ﮔﺮﻓﺘﻪ ﺷﺪه ﺑﻮد‪ .‬اﺳﺘﻔﺎده از ﺷﻴﺐ ﻧﻤﻜـﻲ‪ ،‬ﺳـﺒﺐ اﺛـﺮ‬ ‫در ﻓﺮﻣــﻮل‪ ،‬ﻣﻘــﺎدﻳﺮ ‪ IC50‬ﺑــﻪ ﺗﺮﺗﻴــﺐ ‪ 950/0 ± 0/5‬و ‪250/0 ± 0/7‬‬
‫ﺟﺪاﺳﺎزي ﻛﻨﻨﺪهي ﭘﺮوﺗﺌﻴﻦ از رزﻳﻦ در ﻛﺮوﻣﺎﺗﻮﮔﺮاﻓﻲ ﺗﻌـﻮﻳﺾ ﻳـﻮﻧﻲ‬ ‫ﻣﻴﻜﺮوﮔﺮم‪/‬ﻣﻴﻠﻲﻣﺘﺮ ﺑـﻪ دﺳـﺖ آﻣـﺪ )ﺷـﻜﻞ ‪ .(3‬آﻧـﺎﻟﻴﺰ آﻣـﺎري ﺑﻴـﺎﻧﮕﺮ‬
‫ﻣﻲﮔﺮدد )‪ .(18‬اﺳﺘﻔﺎده از ‪ ،NaCl‬ﺑﻪ دﻟﻴﻞ ﺧﺎﺻﻴﺖ ﺿﻌﻴﻒ ﺟﺪاﺳـﺎزي‬ ‫اﺧﺘﻼف ﻣﻌﻨﻲدار ﻣﻴﺎن اﻳﻦ دو ﻋﺪد ﻣﻲﺑﺎﺷﺪ )‪.(P < 0/010‬‬

‫ﻛﻨﻨﺪه ﺑﻪ ﺳﺎﻳﺮ ﻣﺤﻠﻮلﻫﺎ ﺗﺮﺟﻴﺢ داده ﻣﻲﺷﻮد )‪.(16‬‬

‫ﻧﺘﺎﻳﺞ ﻣﻴﺰان ﻓﻌﺎﻟﻴﺖ ﻣﻬﺎري اﻧﺪازهﮔﻴﺮي ﺷﺪه در ﻣﻄﺎﻟﻌﻪي ﺣﺎﺿـﺮ‪،‬‬ ‫ﺳﺎﻋﺖ‪48h‬‬
‫‪cell 48‬‬ ‫ﺳﻠﻮلﻫﺎ ﭘﺲ از‬
‫‪viability‬‬ ‫درﺻﺪ زﻧﺪه ﻣﺎﻧﺪن ‪%‬‬
‫‪100‬‬
‫‪24h‬‬ ‫‪cell 24‬‬
‫ﺳﺎﻋﺖ ‪ ‬‬ ‫‪viability%‬‬
‫درﺻﺪ زﻧﺪه ﻣﺎﻧﺪن ﺳﻠﻮلﻫﺎ ﭘﺲ از‬
‫درﺻﺪ زﻧﺪه ﻣﺎﻧﺪن ﺳﻠﻮلﻫﺎ‬

‫ﺑﺎ ﻫﻴﭻ ﻣﻄﺎﻟﻌﻪي دﻳﮕﺮي ﻗﺎﺑﻞ ﻣﻘﺎﻳﺴﻪ ﻧﻤﻲﺑﺎﺷـﺪ؛ ﭼـﺮا ﻛـﻪ ﺑـﺮ اﺳـﺎس‬
‫‪80‬‬
‫ﺟﺴﺘﺠﻮﻫﺎي اﻧﺠﺎم ﺷﺪه‪ ،‬ﻣﺸﺨﺺ ﮔﺮدﻳﺪ ﻛﻪ ﺳـﺎﻳﺮ ﻣﻄﺎﻟﻌـﺎت از روش‬
‫‪60‬‬
‫ﻛﻴﻔﻲ زﻳﻤﻮﮔﺮاﻓﻲ )‪ (Zymography‬اﺳﺘﻔﺎده ﻛﺮدهاﻧﺪ )‪ .(9-10 ،12‬ﺗﻨﻬﺎ‬
‫‪40‬‬
‫در ﻣﻄﺎﻟﻌﻪي ‪ Sun‬و ﻫﻤﻜﺎران‪ ،‬ﻓﻌﺎﻟﻴﺖ ﻣﻬـﺎري وﻳـﮋهي اﺳـﭙﻮراﻣﻴﻦ ﺑـﻪ‬ ‫‪20‬‬
‫ﺻﻮرت درﺻﺪي ﮔﺰارش ﺷﺪه اﺳﺖ )‪ ،(11‬اﻣـﺎ در ﺗﺤﻘﻴـﻖ ﺣﺎﺿـﺮ‪ ،‬از‬ ‫‪0‬‬
‫روش ﻣﻌﺘﺒــﺮ و ﻛﻤــﻲ ‪ Laskowaski-Takanara‬اﺳــﺘﻔﺎده ﮔﺮدﻳــﺪ‪ .‬در‬ ‫‪0‬‬ ‫‪200 400 600 800 1000 1200 1400 1600 1800 2000‬‬

‫روش زﻳﻤﻮﮔﺮاﻓﻲ‪ ،‬ﺗﻨﻬﺎ ﻣﻲﺗﻮان ﻧﺸﺎن داد ﻛﻪ اﺳﭙﻮراﻣﻴﻦ ﺧﺎﺻﻴﺖ ﻣﻬـﺎر‬ ‫ﻏﻠﻈﺖ ﻫﺎﺳﭙﻮراﻣﻴﻦ )‪(µg/ml‬‬

‫ﺗﺮﻳﭙﺴﻴﻦ را دارد‪ ،‬اﻣﺎ ﻋﺪدي را ﺑﻪ آن ﻧﺴﺒﺖ ﻧﻤﻲدﻫﺪ‪.‬‬ ‫ﺷﻜﻞ ‪ .3‬درﺻﺪ زﻧﺪه ﻣﺎﻧﺪن ﺳﻠﻮلﻫﺎي‬
‫در ﻧﺘﺎﻳﺞ روش ‪ ،MTT‬ﻣﺸﺨﺺ ﺷﺪ ﻛﻪ اﺛﺮ ﺳﻤﻴﺖ اﺳﭙﻮراﻣﻴﻦ ﺑـﺮ‬ ‫‪ 24 ،(MCF-7) Michigan cancer foundation-7‬و ‪ 48‬ﺳﺎﻋﺖ‬
‫ﺳﻠﻮلﻫﺎي ﺳﺮﻃﺎن ﭘﺴﺘﺎن واﺑﺴﺘﻪ ﺑﻪ دز و زﻣﺎن ﻣﻲﺑﺎﺷﺪ؛ ﺑﻪ ﮔﻮﻧﻪاي ﻛـﻪ‬ ‫ﭘﺲ از ﺗﻴﻤﺎر ﺑﺎ ﻏﻠﻈﺖﻫﺎي ﻣﺘﻔﺎوت اﺳﭙﻮراﻣﻴﻦ‬

‫ﻣﺠﻠﻪ داﻧﺸﻜﺪه ﭘﺰﺷﻜﻲ اﺻﻔﻬﺎن – ﺳﺎل ‪ / 35‬ﺷﻤﺎرهي ‪ /430‬ﻫﻔﺘﻪي دوم ﺗﻴﺮ ‪  1396‬‬ ‫‪568‬‬

‫‪www.mui.ac.ir‬‬
 ‫ﻣﺮﺿﻴﻪ ﻗﻴﻮﻣﻴﺎن و ﻫﻤﻜﺎران‬
‫اﺳﭙﻮراﻣﻴﻦ راﺣﺖﺗﺮ و ارزانﺗﺮ ﺗﻬﻴﻪ ﺷﻮد و ﺑـﺎ ﺗﻮﺟـﻪ ﺑـﻪ ﻣـﺆﺛﺮ ﺑـﻮدن‬
‫ ﻣﻲﺗﻮان ﻣﻄﺎﻟﻌﺎت‬،‫اﺳﭙﻮراﻣﻴﻦ در ﻛﺎﻫﺶ رﺷﺪ ﺳﻠﻮلﻫﺎي ﺳﺮﻃﺎن ﭘﺴﺘﺎن‬
.‫ﺑﻴﺸﺘﺮي ﺑﺮ روي آن اﻧﺠﺎم داد‬
‫ﺗﺸﻜﺮ و ﻗﺪرداﻧﻲ‬
‫ ﺑﺨﺸﻲ از ﻃﺮح ﭘﮋوﻫﺸﻲ ﻣﺼﻮب در ﻣﻌﺎوﻧﺖ ﺗﺤﻘﻴﻘﺎت‬،‫ﻣﻘﺎﻟﻪي ﺣﺎﺿﺮ‬
‫و ﻓــﻦآوري داﻧﺸــﮕﺎه ﻋﻠــﻮم ﭘﺰﺷــﻜﻲ ﮔــﻴﻼن ﺑــﺎ ﺷــﻤﺎرهي ﺛﺒــﺖ‬
‫ از ﺟﻨـﺎب آﻗـﺎي ﻓـﺮحﺑﺨـﺶ‬.‫ ﻣﻲﺑﺎﺷـﺪ‬IR.GUMS.REC.1394.32
‫ ﺑﻪ دﻟﻴـﻞ ﺣﻤﺎﻳـﺖﻫـﺎي ﻓﻨـﻲ و‬،‫ﻛﺎرﺷﻨﺎس ﻣﺤﺘﺮم آزﻣﺎﻳﺸﮕﺎه ﺑﻴﻮﺷﻴﻤﻲ‬
.‫ ﺳﭙﺎﺳﮕﺰاري ﻣﻲﮔﺮدد‬،‫ﻣﻌﻨﻮي‬
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569 1396 ‫ ﻫﻔﺘﻪي دوم ﺗﻴﺮ‬/430 ‫ ﺷﻤﺎرهي‬/ 35 ‫ﻣﺠﻠﻪ داﻧﺸﻜﺪه ﭘﺰﺷﻜﻲ اﺻﻔﻬﺎن – ﺳﺎل‬
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‫ﺑﻬﻴﻨﻪﺳﺎزي ﺗﺨﻠﻴﺺ اﺳﭙﻮراﻣﻴﻦ ﺳﻴﺐزﻣﻴﻨﻲ ﺷﻴﺮﻳﻦ‬
‫ ﻛﺎﻫﺶ ﻣﻲﻳﺎﺑﺪ ﻛـﻪ ﻣﻄـﺎﺑﻖ ﺑـﺎ‬IC50 ،‫ ﺳﺎﻋﺖ‬48 ‫ ﺑﻪ‬24 ‫ﺑﺎ ﮔﺬر زﻣﺎن از‬
‫( و در ﺧﺼـﻮص‬9 ،12) ‫ﻧﺘﺎﻳﺞ ﺳﺎﻳﺮ ﻣﻄﺎﻟﻌﺎت اﻧﺠﺎم ﺷﺪه ﺗﺎﻛﻨﻮن اﺳﺖ‬
‫ ﺗﺄﻛﻴﺪ ﻣﻄﺎﻟﻌﺎت ﻗﺒﻠﻲ ﺑـﺮ ﻣﺴـﻴﺮ ﻣﻴﺘﻮﻛﻨـﺪرﻳﺎﻳﻲ‬،‫ﻣﻜﺎﻧﻴﺴﻢ اﺛﺮ اﺳﭙﻮراﻣﻴﻦ‬
‫ ﺑﺮ اﺳﺎس ﻣﻄﺎﻟﻌﺎت اﻧﺠـﺎم ﮔﺮﻓﺘـﻪ ﺑـﺮ‬.(9-10 ،12) ‫آﭘﻮﭘﺘﻮز ﺑﻮده اﺳﺖ‬
‫ ﺳﺒﺐ اﻓـﺰاﻳﺶ ﺑﻴـﺎن‬،‫ ﺗﻴﻤﺎر ﺑﺎ اﺳﭙﻮراﻣﻴﻦ‬،‫روي ﺳﻠﻮلﻫﺎي ﺳﺮﻃﺎن زﺑﺎن‬
‫( و ﺗﻨﻈـﻴﻢ ﻣﻨﻔـﻲ‬BCL-2) B-cell lymphoma-2 ‫ ﻛﺎﻫﺶ ﺑﻴﺎن‬،Bax
Protein kinase B/Glycogen synthase kinase 3 ‫ﻣﺴــﻴﺮ‬
.(10) ‫( ﻓﺴﻔﺮﻳﻼﺳﻴﻮن ﻣﻲﮔﺮدد‬Akt/GSK3)
‫ﺑﺎ ﺗﻮﺟﻪ ﺑﻪ ﻛﺎراﻳﻲ روش ﭘﻴﺸﻨﻬﺎدي ﺧـﺎﻟﺺﺳـﺎزي اﺳـﭙﻮراﻣﻴﻦ از‬
،‫ ﻣـﻲﺗـﻮان اﻣﻴـﺪوار ﺑـﻮد ﻛـﻪ در آﻳﻨـﺪه‬،‫رﻳﺸﻪي ﺳـﻴﺐزﻣﻴﻨـﻲ ﺷـﻴﺮﻳﻦ‬
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tongue carcinoma cells by down-regulating
Akt/GSK-3 signaling. Fundam Clin Pharmacol 2011;
25(2): 229-36.
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inhibitor activity of a sporamin B from sweet potato
(Ipomoea batatas Lam. 55-2). Agr Sci China 2009;
8(7): 808-20.
12. Huang GJ, Sheu MJ, Chen HJ, Chang YS, Lin YH.
Growth inhibition and induction of apoptosis in NB4
promyelocytic leukemia cells by trypsin inhibitor
from sweet potato storage roots. J Agric Food Chem
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Enzymol 1955; 2: 26-36.
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endogenous metabolic marker for neuronal activity.
Trends Neurosci 1989; 12(3): 94-101.
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 Journal of Isfahan Medical School Received: 28.04.2017
 
Vol. 35, No. 430, 2nd Week, July 2017 Accepted: 17.06.2017

Optimization Sweet Potato [Ipomoea Batats (L.) Lam] Sporamin Extraction and
Analyzing its Antiproliferative Effect on Breast Cancer Cells, MCF-7 Cell Line

Marzyeh Ghayoumian1, Adeleh Alihashemi1, Maryam Omidioskuie2, Iraj Nikokar3, Azadeh Kabiri4

Original Article
Abstract
Background: Finding cures with herbal origins with an easy and cost-effective way of extraction for treatment of
diseases such as cancer is highly appreciated. Considering that proteases are involved in initiation and spreading of
cancer cells, proteases inhibitors can be take into account as an option for treatment. Sporamin is belonging to
trypsine inhibitors group which exists in sweet potato tuber. In this study, the method of sporamin extracting was
optimized, and for the first time, its effect on proliferation of breast cancer, MCF-7 cell line, was examined.
Methods: Aqueous extraction was obtained from sweet potato. Transparent extraction was inserted into
chromatography diethylaminoethanol-Sepharose (DEAE-Sepharose) column. After washing with a linear salt
gradient, fractions were separated. To determine protein purity, sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) was performed. Sporamin inhibitory activity was measured using Laskowaski
method, and eventually 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT method) was used to
assess the antiproliferative effect of sporamin.
Findings: A single band of 25 kDa sporamin was obtained via SDS-PAGE. Its inhibitory effect was measured
800 units in mg/minute. According to the results of MTT method, the amounts of inhibitory concentration 50
(IC50) for 24 and 48 hours were significantly different (P < 0.01).
Conclusion: Using single-column ion exchange and linear salt gradient for sporamin purification was
successful; and due to its antiproliferative effect on MCF-7 cells, there is hope that this technique could be used
for the purification of drug and treatment of disease in future.
Keywords: Ipomoea batatas, Purification, Chromatography, Electrophoresis, Polyacrylamide gel, Breast neoplasms

Citation: Ghayoumian M, Alihashemi A, Omidioskuie M, Nikokar I, Kabiri A. Optimization Sweet Potato

[Ipomoea Batats (L.) Lam] Sporamin Extraction and Analyzing its Antiproliferative Effect on Breast
Cancer Cells, MCF-7 Cell Line. J Isfahan Med Sch 2017; 35(430): 565-70.

1- Medical Biotechnology Research Center, School of Nursing, Midwifery and Paramedicine, Guilan University of Medical Sciences,
Rasht, Iran
2- Instructor, Medical Biotechnology Research Center, School of Nursing, Midwifery and Paramedicine, Guilan University of Medical
Sciences, Rasht, Iran
3- Associate Professor, Medical Biotechnology Research Center, School of Nursing, Midwifery and Paramedicine, Guilan
University of Medical Sciences, Rasht, Iran
4- Assistant Professor, Medical Biotechnology Research Center, School of Nursing, Midwifery and Paramedicine, Guilan
University of Medical Sciences, Rasht, Iran
Corresponding Author: Azadeh Kabiri, Email: kabiri@gums.ac.ir

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