《河南師范大學(xué)-細(xì)胞生物學(xué)-第三章》文檔閱讀

第三章細(xì)胞生物學(xué)研究方法內(nèi)容提要細(xì)胞形態(tài)結(jié)構(gòu)的觀察方法細(xì)胞組分的分析方法細(xì)胞培養(yǎng)、細(xì)胞工程和顯微操作技術(shù)細(xì)胞生物學(xué)研究的模式生物第一節(jié)細(xì)胞形態(tài)結(jié)構(gòu)的觀察方法顯微鏡的出現(xiàn)，結(jié)合細(xì)胞化學(xué)的方法，尤其是免疫細(xì)胞化學(xué)技術(shù)，細(xì)胞整體形態(tài)、細(xì)胞的結(jié)構(gòu)、細(xì)胞中的分子甚至原子都得以揭示。Figure

3-1.

Resolving

power.

Sizeof

cells

and

their

components

drawnon

a

logarithmic

scale,

indicatingthe

range

of

objects

that

can

bereadily

resolved

by

the

naked

eyeand

in

the

light

and

electronmicroscopes.一、光學(xué)顯微鏡技術(shù)：光學(xué)顯微鏡技術(shù)是細(xì)胞生物學(xué)研究常用手段之一。光學(xué)顯微鏡構(gòu)成：光學(xué)放大系統(tǒng)（包括物鏡和目鏡）照明系統(tǒng)支撐系統(tǒng)1

復(fù)式光學(xué)顯微鏡技術(shù)顯微鏡最為重要的性能參數(shù)是分辨率（指區(qū)分開(kāi)兩個(gè)質(zhì)點(diǎn)間的最小距離），而非放大倍數(shù)。性能參數(shù)：分辨率(D值)越小，分辨率越高（D值與光源波長(zhǎng)λ成正比，與介質(zhì)折射率N成反比；所以波長(zhǎng)越短，分辨率越高，介質(zhì)折射率越大，分辨率越高）0.61λN

·

sin（α/2）D=以波長(zhǎng)最短的可見(jiàn)光（400-700nm）的藍(lán)光（450nm）為光源，空氣為折射介質(zhì)，光學(xué)顯微鏡最大分辨率0.3um；以油為折射介質(zhì)，可以達(dá)到0.2um；如果以紫外線（200-300nm）作為光源，分辨率可達(dá)到0.1um。光鏡下可以看到的結(jié)構(gòu)包括細(xì)菌、線粒體（0.5um大小，是光鏡下能清晰可見(jiàn)的最小物體）、中心體、核仁等，稱為顯微結(jié)構(gòu)。組織切片的制備－包埋－切片－脫蠟－復(fù)水－染色－脫水－透固定的目的是使細(xì)胞及其成分鎖定在原有的位置，常用的有乙醇、甲醇、甲醛、戊二醛等。常用的染色劑有蘇木精、伊紅、孔雀綠、蘇丹黑、考馬斯亮藍(lán)等，它們對(duì)細(xì)胞某一特殊的亞細(xì)胞成分有特異的親和性。切片厚度1-10um使用明視野顯微鏡（bright-fieldmicroscope）很難觀察到小的、未經(jīng)染色的樣品，如：活細(xì)胞。相差顯微鏡（phase-contrastmicroscope）則易于觀察高透明的物體。2相差和微分干涉顯微鏡技術(shù)相差顯微鏡：利用光的衍射現(xiàn)象，通過(guò)增加相差版和環(huán)形光闌，將肉眼不能察覺(jué)的相位差轉(zhuǎn)變成振幅差，從而表現(xiàn)為肉眼可見(jiàn)的明暗區(qū)別。樣品不需要染色，優(yōu)點(diǎn)是能觀察無(wú)色、透明的活細(xì)胞中的結(jié)構(gòu)。利用的是不同成分、部分的光衍射系數(shù)不同的特點(diǎn)。細(xì)胞培養(yǎng)中使用的倒置相差顯微鏡，更便于觀察培養(yǎng)物中的活細(xì)胞。微分干涉顯微鏡：利用光的干涉現(xiàn)象，以平面偏振光為光源，適合觀察活細(xì)胞中較大的細(xì)胞器。與錄像裝置結(jié)合，可以觀察和記錄活細(xì)胞中顆粒及細(xì)胞器的運(yùn)動(dòng)，例如：錄像增差顯微鏡技術(shù)（video-enhance

contrast

microscopy）就是將計(jì)算機(jī)輔助系統(tǒng)應(yīng)用于微分干涉顯微鏡，可以觀察微管上顆粒的運(yùn)動(dòng)。Figure

3-11.Four

types

of

light

microscopy.(A)The

image

of

afibroblast

in

culture

obtained

by

the

simple

transmission

of

ligh

the

cell,a

technique

known

as

bright-field

microscopy.(B)phase

contrast

microscopy,(C)Nomarski

differential-interference-co

microscopy,and

(D)dark-field

microscopy（暗視野顯微鏡）.Video-enhance(contrast)

microscopy·Observing

living

specimens;·Greatly

increasethecontrast

of

an

image

so

thatvery

small

objects

become

visible.3熒光顯微鏡技術(shù)

熒光顯微鏡的原理是利用一定波長(zhǎng)的紫外線作為光源來(lái)激發(fā)標(biāo)本中存在的熒光物質(zhì)，使其發(fā)出不同顏色的光而成像。是目前光鏡水平對(duì)蛋白等大分子定性定位研究的重要工具。

包括兩套濾光片，激發(fā)光濾片（只讓能激發(fā)特殊熒光染料的波長(zhǎng)通過(guò)）和阻斷濾片（只允許熒光通過(guò)），在黑暗的背景上，發(fā)出熒光的樣品很容易被觀察到。有些生物體中的天然物質(zhì)受到紫外線照射能夠發(fā)出熒光，有些成分則不發(fā)光或熒光微弱，需要特定的熒光染料染色顯示。常用熒光染料：DAPI(聯(lián)脒基苯吲哚)和碘化丙錠可以染DNA；吖啶橙可以染DNA和RNA；熒光黃（fluorescein）受到藍(lán)光激發(fā)產(chǎn)生強(qiáng)烈的綠色熒光；羅丹明（rhodamine）受到黃綠色光激發(fā)可產(chǎn)生深紅色熒光，分別與抗體耦聯(lián)，可以觀察兩種不同抗原分子在同一細(xì)胞內(nèi)的分布。Figure

3-9.

Fluorescence

microscopy.

Micrographs

of

a

portion

of

the

of

an

early

Drosophila

embryo

in

which

the

microtubules

have

been

labeled

with

an

antibody

coupled

to

fluorescein

(left

panel)

and

the

actin

filaments

have

been

la

with

an

antibody

coupled

to

rhodamine

(middle

panel).

In

addition,

the

chromosome

have

been

labeled

with

a

third

dye

that

fluoresces

only

when

it

binds

to

DNA

(right

panel).

At

this

stage,

all

the

nuclei

of

the

embryo

share

a

common

cytoplasm,

and

tare

in

the

metaphase

stage

of

mitosis.

The

three

micrographs

were

taken

of

the

sam

region

of

a

fixed

embryo

using

three

different

filter

sets

in

the

fluorescence

mic在細(xì)胞生物學(xué)與分子生物學(xué)領(lǐng)域中，來(lái)自水母的綠色熒光蛋白基因常被用作為一個(gè)報(bào)告基因

(reporter

gene)。將GFP基因與待測(cè)基因融合，在細(xì)胞內(nèi)表達(dá)，可

以通過(guò)熒光顯微鏡觀察蛋白在活細(xì)胞內(nèi)動(dòng)態(tài)變化。日本科學(xué)家下村修、美國(guó)科學(xué)家馬丁·沙爾菲和美籍華裔科學(xué)家錢永健因在發(fā)現(xiàn)和研究綠色熒光蛋白方面做出貢獻(xiàn)而獲得2008年諾貝爾化學(xué)獎(jiǎng)。4激光掃描共焦顯微鏡技術(shù)20世紀(jì)50年代后期發(fā)明，多技術(shù)的集合。物鏡和聚光鏡同時(shí)聚焦到同一點(diǎn)上（共焦點(diǎn)），可以自動(dòng)改變觀察的焦平面，通過(guò)“光學(xué)切片”獲得一系列細(xì)胞不同切面上的圖像，經(jīng)疊加后

重構(gòu)樣品的三維結(jié)構(gòu)。比普通熒光顯微鏡成像更清晰，分辨率更高。在研究亞細(xì)胞結(jié)構(gòu)與組分的定位及動(dòng)態(tài)變化應(yīng)用廣泛。Figure

3-8.

Comparison

of

conventional

and

confocal

fluorescemicroscopy.

These

two

micrographs

are

of

the

same

intact

gastrula-stage

Drosembryo

that

has

been

stained

with

a

fluorescent

probe

for

actin

filaments.

Theconventional,

unprocessed

image

(A)

is

blurred

by

the

presence

of

fluorescentstructures

above

and

below

the

plane

of

focus.

In

the

confocal

image

(B),

this

outfocus

information

is

removed,

which

results

in

a

crisp

optical

section

of

the

cellembryo.5熒光共振能量轉(zhuǎn)移技術(shù)檢測(cè)活細(xì)胞中生物大分子納米級(jí)距離和納米級(jí)距離變化的有利工具，可以檢測(cè)兩個(gè)蛋白質(zhì)分子是否存在直接的相互作用。方法是將兩種蛋白分別與CFP（

cyanfluorescentprotein）,YFP（

yellowfluorescent

protein

）融合表達(dá)，通過(guò)檢測(cè)

CFP熒光強(qiáng)度的損失量確定兩個(gè)蛋白是否相互作用。如果發(fā)生能量轉(zhuǎn)移，說(shuō)明兩個(gè)蛋白的距離在

10nm的范圍內(nèi)，一般可以認(rèn)定有相互作用。6熒光漂白恢復(fù)技術(shù)利用熒光分子的耦聯(lián)，檢測(cè)活細(xì)胞表面或內(nèi)部的分子運(yùn)動(dòng)以及在各種結(jié)構(gòu)上分子動(dòng)態(tài)變化率的大小。能夠獲得蛋白質(zhì)運(yùn)動(dòng)的定性結(jié)果，還可以得到擴(kuò)散常數(shù)、蛋白移動(dòng)率等定量信息。圖3-10二、電子顯微鏡技術(shù)分辨率更高，1932年Ruska制成。與光鏡的基本區(qū)別：構(gòu)成上：光源為電子束（小于0.1nm），使用電磁透鏡，鏡筒中要求高真空，圖像通過(guò)熒光屏觀察。分辨率上：最高0.2nm，只能在電子顯微鏡下觀察到的結(jié)構(gòu)稱為亞顯微結(jié)構(gòu)（超微結(jié)構(gòu)）。透射電子顯微鏡（transmission

electron

microscope,TEM）利用透過(guò)樣品的電子形成圖像，在觀察細(xì)胞內(nèi)部結(jié)構(gòu)方面應(yīng)用廣泛。掃描電子顯微鏡（scanning

electron

microscope,SEM）利用樣品表面反彈的電子成像，主要用于觀察物體的表面結(jié)構(gòu)。1透射電子顯微鏡放大倍數(shù)在1000-250000倍之間，分辨極限為3~5埃。TEM電鏡制樣技術(shù)：(1)超薄切片技術(shù):厚度40-50nm固定—包埋—切片—染色Figure

3-19.

Diagram

of

the

copper

grid

used

to

support

the

thisections

of

a

specimen

in

the

transmission

electron

microscopThin

sectionsFigure

3-20.

Electron

micrograph

of

a

root-tip

cell

stained

wiosmium

and

other

heavy

metal

ions.

The

cell

wall,

nucleus,

vacuoles,mitochondria,

endoplasmic

reticulum,

Golgi

apparatus,

and

ribosomes

are

easily

sFigure3-21.

Electron

micrograph

of

a

cell

showing

the

location

of

a

particu

enzyme

(nucleotide

diphosphatase)

in

the

Golgi

apparatus.

A

thin

section

of

twas

incubated

with

a

substrate

that

formed

an

electron-dense

precipitate

upon

rea

with

the

enzymeFigure

3-63.

Immunogold

electron

microscopy.

Electron

micrographsinsulin-secreting

cell

in

which

the

insulin

molecules

have

been

labeled

with

antiantibodies

bound

to

tiny

colloidal

gold

spheres.

Most

of

the

insulin

is

stored

indense

cores

of

secretory

vesicles;

in

addition,

some

cores

are

being

degraded

inlysosomes.(2)冷凍超薄切片技術(shù)可以保存可溶性物質(zhì)和生物大分子的活性（尤其是酶），能夠在分子水平上研究細(xì)胞的超薄結(jié)構(gòu)。樣品放置在液態(tài)丙烷（-42℃）或液氦（-273℃）冷卻的金屬塊上，快速冷凍，冰晶沒(méi)有時(shí)間生長(zhǎng)到明顯破壞細(xì)胞結(jié)構(gòu)的程度。

冷凍切片制備快速，在臨床上用于切除組織的惡性或良性的鑒定。(3)冷凍蝕刻電鏡技術(shù)通過(guò)樣品迅速冷凍和低溫下斷裂，觀察膜斷裂面的蛋白質(zhì)顆粒和膜表面結(jié)構(gòu)。樣品可不經(jīng)包埋和固定處理。Figure.

Freeze-fracture

electron

micrograph

of

the

thylakoid

membranes

frchloroplast

of

a

plantcell.

These

membranes,

which

carry

out

photosynthesis,stacked

up

in

multiple

layers.

The

largest

particles

seen

in

the

membrane

are

thecomplete

photosystem

II-a

complex

of

multiple

proteins.Freeze-Fracture

and

Freeze-Etch

Electron

MicroscopyFigure

3-22.

Three-dimensional

reconstruction

from

serial

seSingle

thin

sections

sometimes

give

misleading

impressions.

In

thexample

most

sections

through

a

cell

containing

a

branchedmitochondrion

will

appear

to

contain

two

or

three

separate

mitochSections

4

and

7,

moreover,

might

be

interpreted

as

showing

a

mitochondrion

in

the

process

of

dividing.

The

true

three-dimensionalhowever,

can

be

reconstructed

from

serial

sections.系列切片的三維重構(gòu)2

掃描電子顯微鏡：主要觀察樣品表面的形貌特征。制樣不需要切片和染色。

為防止樣品在干燥中變形，常用CO2臨界點(diǎn)干燥法，樣品觀察前要用鍍金（一般是金或金-鈀合金）的方法使標(biāo)本導(dǎo)電。

觀察樣品的大小范圍從病毒到昆蟲(chóng)的頭部，一般放大為15-150000倍，分辨率3nm。Figure

3-23.

Scanning

electron

microscopy.

Scanning

electronmicrograph

of

the

stereocilia

projecting

from

a

hair

cell

in

the

iof

a

bullfrog

(A).

For

comparison,

the

same

structure

is

shown

bydifferential-interference-contrast

light

microscopy

(B)

and

by

tsection

electron

microscopy

(C).Figure

3-32.

Cells

in

culture.

Scanning

electron

micrograph

offibroblasts

growing

on

the

plastic

surface

of

a

tissue-culture

di3

掃描隧道顯微鏡（scanning

tunnel

microscpoe,STM）1986年獲諾貝爾物理學(xué)獎(jiǎng)，用于觀察大分子、生物膜和病毒等，在納米水平探索微觀世界物質(zhì)表面形貌。第二節(jié)細(xì)胞組分的分析方法細(xì)胞的分離技術(shù)：分離特定類型細(xì)胞亞細(xì)胞組分的分離技術(shù)：分離特定的細(xì)胞器或 組分細(xì)胞化學(xué)技術(shù)：顯示鑒定大分子免疫細(xì)胞化學(xué)技術(shù)：特異蛋白的定位和定性原位雜交技術(shù)：特定核酸序列的定位和定性放射自顯影技術(shù)：標(biāo)記的生物大分子定位、定 量以及大分子動(dòng)態(tài)示蹤1細(xì)胞的分離技術(shù)：（1）流式細(xì)胞技術(shù)（flow

cytometer,FCM）：從組織中分選相對(duì)純化細(xì)胞的方法，利用了熒光染料耦聯(lián)的抗體標(biāo)記細(xì)胞，通過(guò)FCM分選已標(biāo)記和未標(biāo)記的細(xì)胞，依據(jù)的是細(xì)胞內(nèi)發(fā)熒光的

DNA含量不同，或者細(xì)胞表面熒光強(qiáng)度不同的特點(diǎn)。該技術(shù)廣泛用于生物大分子的定量，細(xì)胞周期分析，細(xì)胞表面抗原、受體、染色體、核質(zhì)比例、活細(xì)胞分類純化等研究中。Figure

3-31.

A

fluorescence-activatcell

sorter.

When

a

cell

passes

througthe

laser

beam,

it

is

monitored

forfluorescence.

Droplets

containingsingle

cells

are

given

a

negative

orpositive

charge,

depending

on

whetherthe

cell

is

fluorescent

or

not.

Thedroplets

are

then

deflected

by

anelectric

field

into

collection

tubesaccording

to

their

charge.

Note

that

thcell

concentration

must

be

adjusted

sothat

most

droplets

contain

no

cells

andflow

to

a

waste

container

together

withany

cell

clumps.

The

same

apparatus

can

also

be

used

to

separatefluorescently

labeled

chromosomesfrom

one

another,

providing

valuablestarting

material

for

the

isolation

andmapping

of

genes.細(xì)胞淘洗（elutration）分離細(xì)胞：將細(xì)胞懸浮在淘洗分離室，細(xì)胞受到方向相反的兩種力的作用，細(xì)胞在分離室中的沉積位置取決于沉降速率（與細(xì)胞大小、形狀和密度有

關(guān)）。該方法的特點(diǎn)是分離速度快，同時(shí)可以分離不同周期和不同時(shí)相的細(xì)胞，可用于細(xì)胞周期、細(xì)胞增殖的研究。密度梯度離心法分離細(xì)胞：通過(guò)離心溶液形成密度梯度，以維持重力的穩(wěn)定性來(lái)抑制對(duì)流。2亞細(xì)胞組分的分離技術(shù)：通常是用低滲、超聲、研磨、勻漿或凍融的方法將細(xì)胞裂解，然后通過(guò)差速離心（differentialcentrifugation）使各種亞組分分開(kāi)。不同組分的沉降率依據(jù)大小和形狀的不同，以沉降系數(shù)(S)表示。通過(guò)密度梯度離心，可以將分子量小的核酸或蛋白質(zhì)進(jìn)一步分離純化出來(lái)。例如：15N標(biāo)記大腸

桿菌DNA，用氯化銫密度梯度離心的方法直接證明了DNA的半保留復(fù)制。The

technique

of

differential

centrifugatioS=(dx/dt)/2x=1

10-13sec.Step-by-step

procedure

for

the

purification

of

organelles

bdifferential

centrifugation.Figure

3-35.

Cell

fractionation

bycentrifugation.

Repeatedcentrifugation

at

progressively

higspeeds

will

fractionate

homogenatescells

into

their

components.

In

genethe

smaller

the

subcellular

componenthe

greater

is

the

centrifugal

forcerequired

to

sediment

it.

Typical

valfor

the

various

centrifugation

stepsreferred

to

in

the

figure

arelow

spee1,000

times

gravity

for

10

minutesmedium

speed:

20,000

times

gravityfor

20

minutes

high

speed:

80,000times

gravity

for

1

hour

very

highspeed:

150,000

times

gravity

for

3hours3細(xì)胞化學(xué)技術(shù)基于細(xì)胞內(nèi)的某些成分可以和某些化學(xué)試劑相結(jié)合或呈特殊反應(yīng)，在細(xì)胞內(nèi)或表面形成有色沉淀物或結(jié)合物而顯示細(xì)胞的結(jié)構(gòu)和成分?！藾NA的顯示：Feulgen反應(yīng)√多糖的顯示：過(guò)碘酸雪夫（PAS）反應(yīng)√脂類的顯示：四氧化鋨或蘇丹III√酶的顯示：通常將新鮮標(biāo)本采用快速冷凍切片，盡可能保持其活性，然后將樣品與相應(yīng)底物進(jìn)行共孵育。不同的酶有不同的顯示方法。4免疫細(xì)胞化學(xué)技術(shù)在細(xì)胞水平研究特定的蛋白質(zhì)的定位和定性，利用抗體的抗原特異性識(shí)別結(jié)合細(xì)胞內(nèi)特定的蛋白質(zhì)，再通過(guò)第二抗體的結(jié)合，使信號(hào)加強(qiáng)。最為敏感的增強(qiáng)信號(hào)的方法是用結(jié)合于第二抗體的酶作為標(biāo)記分子。例如：熒光染料用于熒光顯微鏡、辣根過(guò)氧化物酶用于光鏡或電鏡、膠體金顆粒用于電鏡、堿性磷酸酶用于生化檢測(cè)。常用的有免疫熒光技術(shù)、免疫電鏡技術(shù)，臨床上的利用酶聯(lián)免疫吸附試驗(yàn)（ELISA）的敏感性可以檢測(cè)妊娠或各種感染。Figure

3-64.

Indirect

immunocytochemistry.

The

method

is

verysensitive

because

the

primary

antibody

is

itself

recognized

by

mamolecules

of

the

secondary

antibody.

The

secondary

antibody

iscovalently

coupled

to

a

marker

molecule

that

makes

it

readily

deteCommonly

used

marker

molecules

include

fluorescein

or

rhodaminedyes,

the

enzyme

horseradish

peroxidase

or

colloidal

gold

spheresthe

enzymes

alkaline

phosphatase

or

peroxidase.Figure

3-57.

Visualizing

intracellular

Ca2+

concentrations

using

a

fluorescindicator.

The

branching

tree

of

dendrites

of

the

Purkinje

cell

in

the

cerebellumreceives

more

than

100,000

synapses

from

other

neurons.

The

output

from

the

cell

iconveyed

along

the

single

axon

seen

leaving

the

cell

body

at

the

bottom

of

the

pictThis

image

of

the

intracellular

calcium

concentration

in

a

single

Purkinje

cell

wusing

a

low-light

camera

and

the

Ca2+-sensitive

fluorescent

indictor

fura-2.

Theconcentration

of

free

Ca2+

is

represented

by

different

colors,

red

being

the

higheblue

the

lowest.

(Courtesy

of

D.W.

Tank

et

al.)Figure

3-58.

Fluorescent

analogue

cytochemistry.

Fluorescencemicrograph

of

the

leading

edge

of

a

living

fibroblast

that

has

been

injected

withrhodamine-labeled

tubulin.

The

microtubules

throughout

the

cell

have

incorporatlabeled

tubulin

molecules.

Thus

individual

microtubules

can

be

detected

and

theidynamic

behavior

followed

using

computer-enhanced

imaging,

as

shown

here.Although

the

microtubules

appear

to

be

about

0.25

μm

thick,

this

is

an

optical

effthey

are,

in

reality,

only

one-tenththis

diameter.

(Courtesy

of

P.

Sammeh

and

G.Borisy.)5原位雜交技術(shù)原位雜交（in

situ

hybridization）是采用標(biāo)記的核酸探針通過(guò)分子雜交確定特異核苷酸序列在染色體或細(xì)胞中位置的方法。首先在光鏡水平上發(fā)展起來(lái)，又出現(xiàn)了熒光原位雜交（熒光素標(biāo)記探針在熒光顯微鏡下觀察）、電鏡原位雜交（生物素等小分子標(biāo)記探針，通過(guò)

檢測(cè)抗生物素抗體相連的膠體金顆粒顯示出來(lái)）。Figure

7-20.

In

situ

hybridization

for

RNA

localization

in

tisAutoradiograph

of

a

section

of

a

very

young

Drosophila

embryo

that

has

beensubjected

to

in

situ

hybridization

using

a

radioactive

DNA

probe

complementary

togene

involved

in

segment

development.

The

probe

has

hybridized

to

RNA

in

theembryo,

and

the

pattern

of

autoradiographic

silver

grains

reveals

that

the

RNA

mathe

gene

(ftz)

is

localized

in

alternating

stripes

across

the

embryo

that

are

threcells

wide.

At

this

stage

of

development

(cellular

blastoderm),

the

embryo

contaiabout

6000

cells.

(E.

Hafen

et

al,

Cell

37:833-841,

1984.)Tracing

and

Assaying

Molecules

Inside

CellsDistribution

of

OsAQP

mRNA

in

rice

leaves.a,

Negative

control

without

probe(

10×20);

b,

Hybrdization

withOsAQP

probe（the

enlargement

of

positive

sigal,

10×40）.

GC,guard

cells;

Me,

mesophyll

cells;

Ep,

epidermal

cell.6放射自顯影技術(shù)利用放射性同位素的電離輻射對(duì)乳膠的感光作用，對(duì)細(xì)胞內(nèi)生物大分子進(jìn)行定性、定位與半定量研究的細(xì)胞化學(xué)技術(shù)，能夠?qū)ι锎蠓肿舆M(jìn)行動(dòng)態(tài)研究和追蹤。主要步驟：同位素標(biāo)記的生物大分子前體的摻入和細(xì)胞內(nèi)同位素所在位置的顯示。3H-TdR研究DNA;3H-U研究RNA;35S標(biāo)記的Met和Cys研究蛋白質(zhì)將脈沖示蹤實(shí)驗(yàn)和放射自顯影術(shù)結(jié)合，對(duì)闡明細(xì)胞中的過(guò)程，例如分泌蛋白從內(nèi)質(zhì)網(wǎng)到細(xì)胞外的過(guò)程十分有效。脈沖示蹤是將放射性物質(zhì)以脈沖形式加入樣品，經(jīng)極短暫處理后，將其洗去，以非放射性分子代替。將染色體制作技術(shù)和放射自顯影技術(shù)結(jié)合，可以觀察染色體復(fù)制的動(dòng)態(tài)變化、細(xì)胞周期的測(cè)定、鑒別早復(fù)制或晚復(fù)制的染色體。Figure

3-51.

Electron-microscopic

autoradiography.

The

resul

pulse-chase

experiment

in

which

pancreatic

beta

cells

were

fed

3H-

leucine

for

5

minutes

followed

by

excess

unlabeled

leucine

(the

ch

The

amino

acid

is

largely

incorporated

into

insulin,

which

is

dest

secretion.

After

a

10-minute

chase

the

labeled

protein

has

moved

fthe

rough

ER

to

the

Golgi

stacks

(A),

where

its

position

is

reveale

the

black

silver

grains

in

the

photographic

emulsion.

After

a

furt

minute

chase

the

labeled

protein

is

found

in

electron-dense

secre

granules

(B).(Courtesy

of

L.

Orci,

from

Diabetes

31:538-565)第三節(jié)細(xì)胞培養(yǎng)、細(xì)胞工程和顯微操作技術(shù)一、細(xì)胞培養(yǎng)：屬于生命科學(xué)研究的基本實(shí)驗(yàn)技術(shù)，涉及：原核細(xì)胞培養(yǎng)（細(xì)菌）真核單細(xì)胞培養(yǎng)（酵母）動(dòng)物細(xì)胞培養(yǎng)植物細(xì)胞培養(yǎng)細(xì)胞培養(yǎng)的用途廣泛，不僅可以研究細(xì)胞本身的生物學(xué)特性，還可改變培養(yǎng)條件、用特殊的理化因子和生物因子處理，觀察細(xì)胞在形態(tài)、結(jié)構(gòu)、行為、甚至基因的變化，從而得到細(xì)胞生長(zhǎng)、分化、癌變、凋亡、衰老以及其他的病變規(guī)律。1動(dòng)物細(xì)胞培養(yǎng)任何動(dòng)物細(xì)胞的培養(yǎng)都需從機(jī)體獲取細(xì)胞開(kāi)始，因此體外培養(yǎng)細(xì)胞包括：（1）原代細(xì)胞（primary

culture

cell）從機(jī)體取出后立即培養(yǎng)的細(xì)胞，不同類型的細(xì)胞體外培養(yǎng)的難度差別很大。取材—分散細(xì)胞（酶消化處理和細(xì)胞篩濾過(guò)）---制備單細(xì)胞懸液--提供合適的培養(yǎng)環(huán)境（營(yíng)養(yǎng)、溫度、pH、血清、CO2等）--細(xì)胞貼壁—進(jìn)行有絲分裂—形成細(xì)胞單層(2)傳代細(xì)胞（subculture

cell）適應(yīng)在體外培養(yǎng)條件下持續(xù)傳代培養(yǎng)的細(xì)胞。包括兩種類型：有限細(xì)胞系（finite

cell

line）：體外培養(yǎng)傳代次數(shù)有限的細(xì)胞（約50代）。永生細(xì)胞系（infinite

cell

line）：傳代中發(fā)生遺傳突變，具有了癌細(xì)胞的特點(diǎn)，有可能無(wú)限制的傳代培養(yǎng)下去。體外培養(yǎng)細(xì)胞可以分成貼壁型和懸浮型，一般不能保持體內(nèi)原有的細(xì)胞形態(tài)。貼壁型有兩種基本形態(tài)：成纖維樣細(xì)胞和上皮樣細(xì)胞；懸浮型則呈球形。2植物細(xì)胞培養(yǎng)單倍體細(xì)胞培養(yǎng)：花藥離體培養(yǎng)：雄性生殖細(xì)胞---胚狀體---單倍體植株愈傷組織培養(yǎng)：誘導(dǎo)愈傷組織---分化出芽和根---完整植株原生質(zhì)體培養(yǎng)：植物體細(xì)胞---纖維素酶消化細(xì)胞壁---原生質(zhì)體---培養(yǎng)和誘導(dǎo)分化---植株二、細(xì)胞工程：在細(xì)胞水平的生物工程，涉及的技術(shù)包括：細(xì)胞培養(yǎng)細(xì)胞分化的定向誘導(dǎo)細(xì)胞融合顯微注射（1）細(xì)胞融合與細(xì)胞雜交技術(shù)：細(xì)胞融合：兩個(gè)或多個(gè)細(xì)胞融合成一個(gè)雙核或多核細(xì)胞，不經(jīng)有性生殖過(guò)程而得到雜種細(xì)胞的方法。方法：病毒介導(dǎo)（滅活的仙臺(tái)病毒）化學(xué)介導(dǎo)（聚乙二醇）電融合（高壓電脈沖）基因型不同的細(xì)胞形成的融合細(xì)胞叫做異核融合細(xì)胞，在培養(yǎng)過(guò)程中會(huì)發(fā)生染色體丟失，例如：人鼠雜交細(xì)胞。細(xì)胞融合技術(shù)始于20世紀(jì)50年代末，60年代后期發(fā)明了只讓雜種細(xì)胞存活并傳代的技術(shù)。Figure

3-33.

The

production

of

hybrid

cells.

Human

cells

and

mouse

cells

are

futo

produce

heterocaryons,

which

eventually

form

hybrid

cells.

These

particular

hcells

are

useful

for

mapping

human

genes

on

specific

human

chromosomes

becausemost

of

the

human

chromosomes

are

quickly

lost

in

a

random

manner,

leaving

clonesthat

retain

only

one

or

a

few.

The

hybrid

cells

produced

by

fusing

other

types

of

coften

retain

most

of

their

chromosomes.（2）單克隆抗體技術(shù)：抗體：機(jī)體對(duì)抗原刺激應(yīng)答所產(chǎn)生的免疫球蛋白，對(duì)相應(yīng)的抗原有特異反應(yīng)。是B淋巴細(xì)胞分化來(lái)的漿細(xì)胞合成并分泌的。類型：多克隆抗體：眾多B細(xì)胞對(duì)一種抗原的不同抗原

決定簇應(yīng)答而合成的多種免疫球蛋白，為不均質(zhì)抗體。單克隆抗體：從單克隆雜交瘤細(xì)胞產(chǎn)生的抗體，為同一類或同一亞類的免疫球蛋白，其獨(dú)特型和恒定區(qū)完全相同，具有高度的特異性。單克隆抗體技術(shù)是1975年Milstein和Kohler建立的，并于1984年獲諾貝爾醫(yī)學(xué)和生理學(xué)獎(jiǎng)。原理：B淋巴細(xì)胞的免疫（對(duì)8-12周齡小鼠進(jìn)行抗原注射）--分離免疫后小鼠的脾細(xì)胞—與小鼠骨髓瘤細(xì)胞融合—HAT培養(yǎng)液篩選雜交瘤細(xì)胞—具有穩(wěn)定生長(zhǎng)和抗體分泌功能的雜交瘤細(xì)胞的克隆化—收獲單克隆抗體融合后的雜交瘤細(xì)胞具有親本的特性，既可以分泌抗體，又可以在體外培養(yǎng)或移植到體內(nèi)無(wú)限增殖。Figure

3-65.

Preparation

ofhybridomas

that

secretemonoclonal

antibodies

against

aparticular

antigen