姝ｅ紡娴嬪畾鍓嶅姟蹇呭彇 2-3涓鏈熷樊寮傝緝澶х殑鏍锋湰鍋氶娴嬪畾

娴嬪畾鎰忎箟锛欬/span>

C4H鍙堢О鍙嶅紡鑲夋閰?4-鍗曟哀鍖栭叾锛屽瀛樺湪浜庨珮绛夋鐗┿€侀叺姣嶃€佽弻绫讳腑锛岃閰跺偓鍖栬倝妗傞吀缇熷寲浣滅敤浜х敓 4-棣欒眴閰哥洂锛屾槸鑻笝鐑烽€斿緞涓户 L-鑻笝姘ㄩ吀瑙ｆ皑閰朵箣鍚庣殑**涓叧閿叾銆侟/span>

娴嬪畾鍘熺悊锛欬/span>

C4H鍌寲鑲夋閰稿拰 NADP鐢熸垚 4-棣欒眴閰哥洂鍜 NADPH锛屽湪 340nm涓嬫祴瀹 NADPH鐢熸垚閫熺巼锛屽嵆鍙弽鏄 C4H娲绘€с€侟/span>

闇€鑷鐨勪华鍣ㄥ拰鐢ㄥ搧锛欬/span>

绱鍒嗗厜鍏夊害璁°€佸彴寮忕蹇冩満銆佸彲璋冨紡绉绘恫鍣ㄣ€?mL鐭宠嫳姣旇壊鐨裤€佺爺閽点€佸啺鍜岃捀棣忔按銆侟/span>

璇曞墏鐨勭粍鎴愬拰閰嶅埗锛欬/span>

鎻愬彇娑诧細100mL脳1鐡讹紝4鈩冧繚瀛橈紱

璇曞墏涓€锛氭恫浣 60 mL脳1鐡讹紝 4鈩冧繚瀛橈紱

璇曞墏浜岋細绮夊墏脳2鐡讹紝-20鈩冧繚瀛橈紱

绮楅叾娑叉彁鍙栵細

缁嗚弻鎴栧煿鍏荤粏鑳烇細鍏堟敹闆嗙粏鑿屾垨缁嗚優鍒扮蹇冪鍐咃紝绂诲績鍚庡純涓婃竻锛涙寜鐓х粏鑿屾垨缁嗚優鏁伴噺(10⁴涓?锛氭彁鍙栨恫浣撶Н(mL)涓 500锝?000锛?鐨勬瘮渚?寤鸿 500涓囩粏鑿屾垨缁嗚優鍔犲叆 1mL鎻愬彇娑?锛岃秴澹版尝鐮寸缁嗚弻鎴栫粏鑳?鍐版荡锛屽姛鐜 20%鎴 200W锛岃秴澹 3s锛岄棿闅 10s锛岄噸澶 30娆?锛?000g 4鈩冪蹇 10min锛屽彇涓婃竻锛岀疆鍐颁笂寰呮祴銆侟/span>

缁勭粐锛氭寜鐓х粍缁囪川閲?g)锛氭彁鍙栨恫浣撶Н(mL)涓 1锛?锝?0鐨勬瘮渚?寤鸿绉板彇绾 0.1g缁勭粐锛屽姞鍏 1mL鎻愬彇娑?锛岃繘琛屽啺娴村寑娴嗐€?000g 4鈩冪蹇 10min锛屽彇涓婃竻锛岀疆鍐颁笂寰呮祴銆侟/span>

娴嬪畾姝ラ锛欬/strong>

1銆佸垎鍏夊厜搴﹁鎴栭叾鏍囦华棰勭儹 30min浠ヤ笂锛岃皟鑺傛尝闀胯嚦 340nm锛岃捀棣忔按璋冮浂銆侟/span>

2銆佹牱鏈祴瀹欬/span>

(1)鍙栬瘯鍓備簩涓€鐡讹紝鍔犲叆 25mL璇曞墏涓€鍏呭垎婧惰В娣峰寑锛岀疆浜 37鈩?鍝轰钩鍔ㄧ墿)鎴 25鈩?鍏跺畠鐗╃)姘存荡 5min锛涚幇閰嶇幇鐢?閰嶅ソ鍚 24h鍐呯敤瀹?锛汓/span>

(2)鍦 1mL鐭宠嫳姣旇壊鐨夸腑鍔犲叆 50渭L鏍锋湰鍜 950渭L璇曞墏浜岋紝娣峰寑锛岀珛鍗宠褰 340nm澶勫垵濮嬪惛鍏夊€ A1鍜 5min鍚庣殑鍚稿厜鍊 A2锛岃绠 螖A=A2-A1銆侟/span>

C4H娲绘€ц绠楋細

(1)鎸夋牱鏈泲鐧芥祿搴﹁绠楋細

鍗曚綅鐨勫畾涔夛細姣 mg缁勭粐铔嬬櫧姣忓垎閽熶骇鐢 1 nmol鐨 NADPH瀹氫箟涓轰竴涓叾娲诲姏鍗曚綅銆侟/span>

C4H(nmol/min/mg prot)锛漑螖A脳V鍙嶆€幻?蔚脳d)脳10⁹]梅(V鏍访桟pr)梅T=643脳螖A梅Cpr

(2)鎸夋牱鏈矞閲嶈绠楋細

鍗曚綅鐨勫畾涔夛細姣 g缁勭粐姣忓垎閽熶骇鐢 1 nmol NADPH瀹氫箟涓轰竴涓叾娲诲姏鍗曚綅銆侟/span>

C4H(nmol/min/g椴滈噸)锛漑螖A脳V鍙嶆€幻?蔚脳d)脳10⁹]梅(W 脳V鏍访稸鏍锋€?梅T=643脳螖A梅W

(3)鎸夌粏鑿屾垨缁嗚優瀵嗗害璁＄畻锛欬/span>

鍗曚綅鐨勫畾涔夛細姣 1涓囦釜缁嗚弻鎴栫粏鑳炴瘡鍒嗛挓浜х敓 1 nmol NADPH瀹氫箟涓轰竴涓叾娲诲姏鍗曚綅銆侟/span>

C4H(nmol/min/10⁴cell)锛漑螖A脳V鍙嶆€幻?蔚脳d)脳10⁹]梅(500脳V鏍访稸鏍锋€?梅T=1.286脳螖A

V鍙嶆€伙細鍙嶅簲浣撶郴鎬讳綋绉紝1脳10^-3L锛浳碉細NADPH鎽╁皵娑堝厜绯绘暟锛?.22脳10³L/ mol/cm锛沝锛氭瘮鑹茬毧鍏夊緞锛?cm锛沄鏍凤細鍔犲叆鏍锋湰浣撶Н锛?.05 mL锛沄鏍锋€伙細鍔犲叆鎻愬彇娑蹭綋绉紝1 mL锛汿锛氬弽搴旀椂闂达紝5 min锛汣pr锛氭牱鏈泲鐧借川娴撳害锛宮g/mL锛沇锛氭牱鏈川閲忥紝g锛?00锛氱粏鑿屾垨缁嗚優鎬绘暟锛?00涓囥€侟/span>